Laboratory Manual for Chem4411 Final 072109

7/31/2019 Laboratory Manual for Chem4411 Final 072109

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Laboratory Manual for

CHEM4411

Fall 2009

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This manual was prepared by the collaborative efforts of the University of Virginia

Chemistry Department graduate and undergraduate students. Lauren Lee and Ana

Wang developed and tested many of the protocols so that each one could be

efficiently conducted in an undergraduate teaching laboratory. Daniel Fox, Ling

Huang, Tomasz Kabsinski, Brett Kroncke, Jenny Lounsbury, William Peairs, and

Brian Poe prepared this manual and improved upon the protocols to enable the

students to obtain meaningful results. In addition, these students worked together

to transform the biochemistry laboratories into a productive and fun space.

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Table of Contents

Syllabus4

Laboratory Station Contents.......9

Summary of Lab Reports...10

Laboratory 1...11

Laboratory 2...18

Laboratory 3...22

Laboratory 4...32

Laboratory 5...43

Laboratory 6...49

Laboratory 7...55

Laboratory 8...63

Laboratory 9...69

Laboratory 10.80

Appendix I: Useful websites .....89

Appendix III: Phosphate Buffer Table...90

Appendix III: Graphing calculators...91

Appendix IV: SDS-PAGE molecular weight standards92

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4

CHEM4411 Biological Chemistry Lab I

This course is designed to give you a glimpse of the general methods utilized in a biochemistrylaboratory. You will perform techniques such as chromatography, PCR, SDS-PAGE gel

electrophoresis, and many more. The lecture each week will address the method, data, and

interpretation of the results for each week. The answers will not be given to you. You will need

to perform literature searches and dig for relevant data in the literature to understand andcompare to your data. This is all meant to provide you with the tools to conduct research, both in

and out of the lab.

Meeting Times and Places

Lecture Monday 2 2:50 p.m. CHM304

Lab Tuesday, Wednesday, or Thursday 2 6:00 p.m. CHM315 or 412

Office Hours

TAs: Monday 3 p.m. 4th

floor hallway/computer roomFriday 10 a.m. 4

thfloor hallway/computer room

Prof. Columbus: Monday 4 p.m.

Required materials

Lab Manual: Available at the bookstore.Lab Notebook: Buy the type with carbon capabilities and duplicate numbered pages.

Text: Fundamental Laboratory Approaches for Biochemistry and Biotechnologyby Ninfa,

Ballou, and Benore.

Comments about Biochemistry Laboratory Protocols

All of these experiments work. The results may not be what you expect, and interpretation ofyour data is not necessarily straightforward. If you dont obtain good results, there are sample

data available that have been obtained by the protocols provided to you. If you need to use the

sample data, then you need to discuss what you did wrong and what could be improved. It is notenough to just do the protocol given to you. You must understand why you are doing a particular

procedure and what the purpose of each step is. There are particular labs that require you to come

in the evening before and the morning of your laboratory. Plan ahead.

Biochemistry laboratory does not usually work in a set four hour period. There will be a lot of

waiting time for certain labs. Bring work with you so that the down time is not wasted time.Also, there are many labs that will go over time; you may want to work out a system with your

lab partner so that you can alternate staying later.


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Syllabus

Week Date Lecture Reading Material Topic

1 8/25 No lecture No labs

2 8/31 No lecture No labs

3 9/7 Introduction to the course Chapter 1 & 2Lab 1: Chec

general lab

4 9/14 Buffers and solutions Chapters 1 & 2 Lab 2: Buff

5 9/21 DNA: Experimental methods Chapter 14 Lab 3: DNA

6 9/28 Proteins: detection and quantification Chapter 3 & 4Lab 4: Prot

determinati

7 10/5 No lecture Reading Days No labs

8 10/12 Cloning Chapter 13Lab 5: Gene

recombinan

9 10/19Recombinant protein expression and

interpreting an SDS-PAGE gelChapter 6

Lab 6: Reco

and SDS-PA

10 10/26 Chromatography I Chapter 5, 7 & 8 Lab 7: Gel

11 11/2 Chromatography II Chapter 5, 7 & 8 Lab 8: Ion e

12 11/9 Chromatography III Chapter 5, 7 & 8 Lab 9: Affin

13 11/16 Enzyme Kinetics Chapter 10 Lab 10: Lac

14 11/23 No lecture Happy Thanksgiving No labs

15 11/30 No lecture Check-out

16 12/7 Q & A session to review for the exam


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Laboratory Sessions

Laboratory sessions begin promptly at 2 p.m. and conclude at or before 6 p.m. You are expectedto read the lab handout before the beginning of the laboratory session, and you may be required

to submit a pre-lab at the start of the laboratory period that will be graded. If you are prepared for

lab, you should have no difficulty completing the experiments in the allotted time. You must

wash your glassware and clean your station after every lab. You risk point deductions from yournext lab report if your TA sees any lab misconduct or messes.

Treat your TA with the utmost respect. If you are frustrated, then it is likely your TA is as well.

State any concerns in clear and respectful language. Refrain from yelling, complaining, or

whining because this will only exacerbate the problem.

Lab Lecture

A one hour laboratory lecture will be given on Monday at 2 p.m. in Room 304 Chemistry. This

period will be used to discuss principles demonstrated during the laboratory sessions as well asadditional methodologies relevant to biochemical research.

Lab Notebooks

The laboratory notebook is an extremely useful tool for record-keeping and is essential for

accurate performance in the laboratory. The notebook must be a permanently bound record book.

All records must be kept in permanent ink. Neatness in the notebook is critical to laboratorytechnique. The notebook should not only be intelligible to the student, but also to any trained

analyst who could repeat the work or complete an unfinished analysis.

Original data must not be altered by erasing or using correction fluid. If an error incalculations or data observations is made, correct the data by drawing a single line through it. Be

sure to explain why the data was excluded.

The record book should also contain a table of contents and numbered pages. The date performed

and initials should also appear on each data page. Observations made during the course of anexperiment should be recorded to help interpret results. Sign and date each page in the notebook.

Enter data in a clear and organized manner. It may be useful to set up the data page beforecollecting data.

Clearly label all entries including units.

Fill in the data in chronological order.

When instruments are used, record the brand, model number, and serial number. It is alsoimportant to record dial settings for any conditions on the instrument which can be

changed.

Affix all graphs, spectra, etc., in the notebook.

Show at least one calculation for each manipulation involved in your calculations.

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Pre-lab Write-upsYou must come to lab prepared with your lab notebook, completed pre-lab, calculator, etc. The

more prepared you are, the smoother lab will go. If you do not have your notebook, you will not

be able to complete the lab. Your pre-lab write-up should be in your notebook.

Pre-labs must be written neatly in your notebook. Even though this portion is not graded, I will

check and initial your notebook each week to be sure the pre-lab is completed. If your pre-lab isnot done, you may not be allowed to complete the lab. The pre-labs help you prepare for lab. If

you complete them, the quizzes should be no problem for you. Pre-labs should include the

following:

1. Procedure: A timeline of tasks to be completed in this lab. Estimate the time it will take todo each task to the best of your knowledge and in what order you should do each task.

Remember: You do not have to proceed in the same order listed in the instructions unlessotherwise stated.

2. Reagents: List and estimate the amounts of each reagent you will need during the lab.3. Equations/Calculations: List any equations needed for each lab and perform as many of the

calculations as possible before you come to lab. For example, concentration and dilutioncalculations will come in handy if they are done beforehand.

Pre-lab quizzes

There will be a brief quiz (five minutes) every week before the start of lab. This quiz will be anassessment of how well you are prepared for lab and will cover the reading material and pre-labs.

You cannot start lab until youve completed and handed in your quiz.

Lab Partners

Lab partners will be assigned before Lab 2 and these assignments will be permanent for theremainder of the semester, unless there is a problem. Please note your partner on both the pre-lab

and your lab reports.

Lab Stations

You will also be assigned a lab station for the remainder of the semester. It is important that you

do not use supplies from other lab stations, even if that station is empty. The contents of yourstation are listed on page 9.

Problems in Lab

If you encounter problems with the equipment, including the pipettes, notify your TAimmediately so he or she can attempt to fix the problem.

Laboratory Safety and Waste Policies

All students must follow safe laboratory practices (http://ehs.virginia.edu/home.html) adopted by

the University of Virginia. You will not be allowed to be in the laboratory with open-toed shoes,

skirts, or shorts. Goggles are absolutely required. You will get one warning to put your goggles

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on. After that you will lose five points off your pre-lab quiz for each time that I have to remind

you. Gloves may be required for certain experiments, though it is a good idea to wear them allthe time anyway. Lab coats are not necessary unless otherwise stated.

Before leaving LabAfter you finish your experiment, please make sure to:

a. Clean your station (e.g., refill pipette tips and distilled H2O bottles)b. Empty waste into appropriate containersc. Check your station before you leave to be sure your station is the way you found it. If you

leave before your TA checks your station, you will be considered absent and receive a

zero for the lab.d. Hand in the carbon copy (yellow or blue sheet) of your pre-lab/data to your TA before

you leave.

Honor Requirements

You are encouraged to work with your lab partners during the laboratory session. After youleave the laboratory you are expected to analyze and write up your data individually. All labreports, assignments, and exams should be pledged in accord with the UVA honor system.

Grading

Lab Reports 650 pts

Pre-lab quizzes 200 pts

Final 350 pts___________

1200 pts

The averages of each lab section will be compared and normalized for differences in grading.

Final Exam date and time will be announced in class and posted on Collab.

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Lab Station Contents

o Pipetteso P20o P200o P1000

o Sharpieo Rulero Pipette tips (full)

o 200 Lo 1000 L

o Water bottle (full)o Ice bucketo Beakers (plastic or glass)

o 1 Lo 600 mLo 250 mLo 100 mL

o Erlenmeyer flasks (plastic or glass)o 500 mL (3)o 125 mLo 50 mLo 25 mL

o Test tube trayo Eppendorf tube trayo

Magnetic stir bars (4)o Pipette bulbs (2)

o Graduated cylinderso 1 Lo 500 mLo 250 mLo 100 mLo 25 mL

o Chromatography columnso 1.5 x 20 cm (blue)o 1.5 x 15 cm (yellow)o 0.5 x 10 cm (skinny blue)

o Goggles (2)o Timero Stop-cocko Spatulao Scoopulao Clampso Centrifuge tubes

o 50 mLo 250 mL

o Bottles with capso 1 Lo 500 mL (2)o 250 mLo

125 mL


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Summary of Lab Reports

Lab reports are to be written up individually, not with your lab partner. However, keep in mind

that you and your lab partner have the same data and therefore, should have the same results aftercalculations are completed. You may check with your lab partner in this respect ONLY. You

each may reach different conclusions, which is perfectly acceptablejust be sure to make astrong argument for your conclusion. Lab reports should be typed and pledged. Please refer to

the report write-up information sheet for the proper format and length.Lab reports are due by 2:15 p.m. on the dates listed below (the day of your laboratory

session) and handed directly to your TA. Any reports turned in after the due date will receive

15% off the final grade for each day that it is late.

Lab(s) Due Date

1 (75 pts) 9/15 9/17 - tables, graphs, and/or questions2 (25 pts) 9/22 9/24 - tables, graphs, and/or questions

3 (75 pts) 9/29 10/1 - lab report

4 (100 pts) 10/13 10/15 - lab report5-9 (250 pts) 11/17 11/19 - lab report10 (125 pts) 12/1 12/3 - lab report

The TAs will try to return quizzes and lab reports to you within one week.

There are four formal lab reports and guidelines are provided for each at the end of each section.

Laboratories 5 9 will be all included into one large report. You will want to plan ahead andprepare the data after each week. Only data will be turned in for Laboratories 1 and 2.

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Laboratory 1: Introduction and Pipette Fundamentals

I. Introduction

In Chemistry 451, we will be using micro-pipettes for all of the experiments. These devices areexpensive and somewhat delicate. In order to obtain accurate and precise data, correct operation

of the micro-pipettes is imperative. For this reason, we are going to start the course with an

exercise to familiarize everyone with the micro-pipettes.

Use of the Microliter PipettorA microliter pipettor is a variable-stroke piston pipette. The volume indicator consists of threenumber dials and is read from top to bottom. The three digits indicate the volume selected and

are colored black or red. The black digits on the P-20 and P-200 show microliters and the red

digits on the P-20 show tenths of microliters. For the P-1000, the digits in red represent

milliliters and the digits in black represent microliters. (These details become more obviouswhen the micro-pipettor is in hand.) The range of each pipette is given below. Do not use

outside of these ranges!

Manufacturers Specifications

Model Range, L Accuracy* Precision*

P-20 2-20 1% 0.5%P-200 20-200 0.8% 0.25%

P-1000 200-1000 0.8% 0.2%

*Relative % at mid-range

Accuracy is the closeness to which the dispensed volume approximates the true volume as set on

the pipette. Accuracy is expressed as mean erroror% error, the percent by which the mean

value of a large number of replicate measurements of the same volume will deviate from the

expected or true volume. The accuracy of these pipettes is determined by the factorycalibration and checked gravimetrically using distilled water and an analytical balance. Careful

use will maintain this calibration and accuracy throughout the semester.

Precision refers to the scatter of individual measurements around the mean of replicate

measurements. It can be expressed assample standard deviation.

Operation of the Microliter Pipette1. Set the volume by turning the volume adjustment knob at the end of the pipette until the

correct volume shows on the indicator.

Note: Never go above or below the range of the pipettor! Know these ranges at alltimes.

2. Attach a new disposable tip to the pipette shaft. Press firmly with a slight twisting motion.Make sure you are using tips of the correct size for each pipette.

3. When gathering sample, press the plunger to the first stop. This part of the stroke is thecalibrated volume displayed on the digital volume indicator. Do not press the plunger allthe way down, or you will draw up too much solution.

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4. Holding the microliter pipettor vertically, immerse part of the disposable tip into thesample.

5. Allow the push-button to returnslowly to the up position. Never let it snap up! (If it doeshappen, tell a TA so that the microliter pipettor may be dismantled and cleaned to prevent

corrosion and the contamination of your succeeding samples.) Do this slowly and keep the

tip submerged in the solution to prevent any air bubbles from entering the tipthis willmess up your volume measurement.

6. Wait a few seconds to ensure that the full volume of sample is drawn into the tip.7. Withdraw the tip from the sample liquid. If any liquid remains on the outside of the tip,

wipe it off carefully with a lint-free tissue, taking care not to touch the orifice. You should

observe the liquid in each type of tip with each pipettor so that you can become aware if

there is a significant problem with the pipettor. This is an incredibly important part of the

technique and becoming efficient at pipetting small volumes.8. To dispense the sample, touch the tip end to the sidewall of the receiving vessel and

depress the plungerslowly to the first stop. Wait two seconds. Then press the plunger to the

second stop (the bottom stroke), expelling any residual liquid in the tip.

9.

With the plunger fully depressed, withdraw the microliter pipettor from the vesselcarefully, with the tip sliding along the wall of the vessel.10.Allow the plunger to returnslowly to the up position.11.Discard the tip. You want to use a different tip each time you are gathering/dispensing

different materials. If you dont do this in this lab, your concentrations of solutions will beinaccurate, and as a result, so will your data.

Note: To prevent liquids from being drawn into the microliter pipettor shaft pipette

slowly and never invert or lay microliter pipettor on its side with liquid in the tip.

Refer page 16 in Boyer for more information and pictures.

II. Required Reading

This entire handout Chapters 1 & 2 of Fundamental Laboratory Approaches for Biochemistry and

Biotechnologyby Ninfa, Ballou, and Benore.

III. Pre-Lab

List of reagents Calculate the amount of CoCl2 (H2O)6 needed to make the stock 2 M solution Calculate the concentration of each of the solutions to be analyzed

IV. Materials

Weigh dish Balance P20, P200, and P1000 pipettes Distilled H2O Pipette tips 6 cuvettes 6 test tubes Spectrophotometer

CoCl2 (H2O)6 Bunsen burner and striker Test tube rack Stirring rod Weigh paper Balance Sharpie marker


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IV. Procedure

Part 1 - Introduction to MicroliterPipettingUnderstanding the Limits

Please fill in the following table:

Pipette

lowlimit

(L)

highlimit

(L)

P20

P200

P1000

What would you use?

Please fill in the following table with the most appropriate equipment to measure the listed

volume. (There may be more than one answer for some.)

Volume

Required (L)

Type

(P1000, P200, P20, or

other)

Reading on

Pipette

1 25

2 12.5

3 300

4 5

5 1000

6 958

7 150.2

8 1.5

9 7000

10 1250

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Part 2: Calibrating and Using a Micropipette and the Mass of Water

1. Place a weigh dish on a balance and tare it.2. Pipette 15 L using the correct model of pipette 10 times (a total of 150 L) into the weigh

dish, and record the mass. Do this three times.

3. To save time and materials, just tare the balance between each addition of 150 L of water.Make sure that the balance shows 0.000g before adding any additional water.4. Using the correct model of pipette, find the mass of 50 L of water. Do this three times,and record your measurements. Repeat this step with 250 L and 750 L.

5. Fill out the table below.6. Record these values and determine the average and standard deviation. If your value is

accurate and precise as determined by the TA standard values, you will have successfully

completed the exercise. If not, you will need to do the exercise again to ensure you areprepared to proceed with the course. Pipetting accurately and precisely is a major

component to getting good data in this course.

Observed Mass (mg)

Volume 10 x 15 L 50 L 250 L 750 L

1

2

3

Mean ( )

Std Dev ( )

Low Value (x - )

High Value (x + )

Range ( high - low )

Standard deviation can be calculated with the following equation:

=sqrt ((x - x )2/N)

where N is the number of values.

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Part 3: Pipetting and Dilution Techniques

Separate Dilutions1. Turn on spectrophotometer, as it takes a while to heat up the lamp. The power switch can

be found on left side towards the bottom.

2. Weigh out the appropriate amount of cobalt chloride and make 2 mL of 2.0 M aqueousCoCl2 (H2O)6 (MW = 237.93 g/mol) in a test tube. You may need to heat the solutionwith a Bunsen burner to make sure it all dissolves. Do not keep the solution over the

flame for an extended amount of time, as it will boil over and burn. Stir the solution witha stirring rod, and make sure that it does not look dusty in the lightthis will affect

your absorbance readings.

3. While taking note of the recommended range of each pipette model and using the correctsize tips, make the following solutions in six separate test tubes. (Be sure to label them soyou dont mix them up!)

Tube Distilled H2O (L) 2M Cobalt Chloride (L) Concentration (M)

1 1000 - 0

2 985 153 975 25

4 800 200

5 700 300

4. Look at the level of solution in each test tube. If your pipetting was accurate, each of thetest tubes should have the same amount of solution. Mix the solutions well using astirring rod.

5. Using the correct pipette, transfer each of the solutions from the test tubes to separatecuvettes. When handling the cuvettes, try not to touch the sides, as smudges on them candisrupt your absorbance measurements.

6. In sample slots of the spectrophotometer, place cuvettes in the following order from theslot nearest to you to the slot farthest from you: 1, 2, 3, 4, 5. Take careful note of what

direction they should be placed in the slot. (Hint: The light beam of thespectrophotometer is horizontal!) Close the compartment.

7. On the spectrophotometer, select the first option 1. ABS/%T/CONC.8. By pressing CELL with the up and down arrows on the keypad, adjust the cuvette of

interest to cuvette 1 (the blank). The screen should have this slot listed as B.

9. Press Go to WL and set the wavelength to 510 nm. Press enter.10.Press the Auto zero button, and make sure that the absorbance reading for your blank is

listed at 0.00A before proceeding.

11.Pressing the up and down CELL key allows the light beam to shine on the differentcuvettes. Press the up CELL key to move onto your solution in slot 2, and record the

absorbance displayed on the screen.12.Do this for each of the solutions, recording their respective absorbencies.13.Make a plot of absorbance vs. concentration. If diluted with proper pipette usage, you

will see a straight line.

Serial Dilutions

1. Label five 13 x 1000 mm test tubes 1-5 (tube 1 will be your blank) and pipette 1000 Lof distilled water into each one.

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2. Add 1000 L of the stock cobalt chloride solution to Tube2. Vortex the solution briefly.3. Pipette 1000 L of Tube2 into Tube3. Vortex briefly.4. Pipette 1000 L of Tube3 into Tube4. Vortex briefly.5. Pipette 1000 L of Tube4 into Tube5. Vortex briefly.6. Remove and discard 1000 L from Tube 5. Check the level of the solution in all your test

tubes. If your serial dilution was done properly, you will have five tests tubes all with thesame amount of solution in them (1000 L).

Tube Concentration (M)

1 0

2

3

4

5

7. Transfer the contents of each tube into 1.5 mL plastic cuvettes.8. Insert the cuvettes into the slots in the instrument, placing your blank cuvette in the first

slot, Tube 2 cuvette in the second slot, and so on, and close the compartment.

9. Continue as before, starting at Step 7 and using the same wavelength (510 nm), untilyouve measured and recorded absorbencies for all your cuvettes.

10.Make a plot of absorbance vs. concentration with your four data points. If diluted withproper pipette usage, you will see a straight line.

Note: Cobalt chloride goes in aqueous waste!

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Data to be turned in for Laboratory 1 (75 pts)

1. The tables from Part 1 and 2, typed or handwritten in pen (carbon copy from your notebook is

fine).

2. Plots of concentration vs. absorbance for each type of dilution (part 3). Label axes and includeunits. Find the equation of the line, and include R-squared values for the linear fit to the data

points. Each plot should have a title and a one-sentence description. This should be typed andeach figure and accompanying text on separate pages (two pages total).

3. Include one sample calculation foreach calculation that you needed for this lab. (Example:How did you calculate concentration of CoCl2 (H2O)6 for each of the dilutions?)

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Laboratory 2: Buffers and Solution Preparation

I. Introduction

Buffers are weak acids and bases. For the dissociation of a weak acid, ,where Ka

is the dissociation constant, we can write:

++ HAHA

][

]][[

HA

AHK

HA

AHa

+

==+

If we are in the dilute limit, then the activity coefficients () approach 1, and we may write:

][

][log

HA

ApKpH a

+=

The above equation is known as the Henderson-Hasselbach Equation. It is an ideal equation,howeverbuffers may perform differently than predicted based on their pKa. These include

variations in the pH of a buffer as a function of both buffer concentration and temperature.Buffers that are called Goods buffers do not have a strong concentration or temperature

dependence and tend to be very compatible with proteins and other biological macromolecules.

By differentiating the above expressions, we can derive an equation for the buffer capacity, ,

which is given by:

2])[(

][3.2+

+

+=

HK

CHK

a

a

where C is the total buffer concentration (C = HA + A-). represents the molar concentration of

H+

that must be added to a solution to produce a single unit change in pH. The higher, the

better the buffer. If we set the derivative of with respect to H+ concentration equal to 0, we canshow that is a maximum when the pH=pKa, and that

max=0.575C.

General Rules for Using Buffers:1) Keep the pH within 1 pH unit of the pKa.

2) Make the buffer up and set its pH close to the working concentration you will use.

3) If you are not using your solution at room temperature, consider the temperature dependenceof the buffer.


This handout Chapter 2 ofFundamental Laboratory Approaches for Biochemistry and Biotechnology

by Ninfa, Ballou, and Benore.

III. Pre-LabFor each of the solutions in the list below, determine which chemical you need and how much

you will need to weigh out. You will need to use the list of available chemicals below, making

careful note of those with similar names.

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Note: These are the buffers and solutions that you will need for the remainder of the

course.

Stock Solutions to Prepare:

a.) 0.5 L of 0.2 M monobasic sodium phosphate (A)b.) 0.1 L of 0.2 M dibasic sodium phosphate (B) (You will more for Lab 10; however,

precipitation is observed over time so you will need to make it fresh.)c.) 100 mL of 1 M Tris base pH 8

d.) 250 mL of 4 M NaCl

e.) 20 mL of 4 M imidazole

f.) 10 mL of 0.5 M MgCl2

Available Chemicals: Listed as: Name (Manufacturer, Formula Weight)

Chemicals in Room 315:

- Imidazole (Acros, FW 68.08)

- Magnesium Chloride Anhydrous (Sigma, FW 95.21)- Magnesium Chloride Hexahydrate (Sigma, FW 203.3)- Sodium Chloride (Sigma, FW 58.44)

- Sodium Phosphate Dibasic Anhydrous (Mallinckrodt, FW 141.96)

- Sodium Phosphate Monobasic Monohydrate (EM Science, FW 137.99)- Sodium Phosphate Monobasic Monohydrate (Sigma, FW 138.0)

- Tris Base (Fisher Bio, FW 121.14)

Chemicals in Room 412:

- Imidazole (Acros, FW 68.08)

- Magnesium Chloride Hexahydrate (Fisher Chem, FW 203.3)- Sodium Chloride (Fisher Chem, FW 58.44)

- Sodium Phosphate Dibasic Anhydrous (Fisher Chem, FW 141.96)

- Sodium Phosphate Monobasic Anhydrous (Sigma, FW 120.0)- Sodium Phosphate Monobasic Monohydrate (EM Science, FW 137.99)

- Tris Base (Fisher Bio, FW 121.14)

Estimate Buffer pH AdjustmentUse the Henderson Hasselbach equation to calculate how much acid you will need to use to

adjust the pH of the Tris base (pKa = 8.3) to pH 8. There are 5 and 1 N HCl solutions available inthe laboratory.

You will be turning in your carbon copies instead of writing a report. Please see the list of

required content after the protocol.

IV. Materials

Beakers Graduated cylinders Distilled H2O Spatulas

pH meter Balance Chemicals listed above


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V. Procedure

Tips/Notes:1. The entire semesters worth of experiments relies on you making accurate buffers;

therefore, take this lab seriously.

2. Check the standards and be sure the pH meter is calibrated each time you use it.3. One of the sodium phosphate salts takes awhile to dissolve; be patient.

4. Be sure to rinse your glassware thoroughly between buffers. It is not recommended to usesoap, which is difficult to remove and can interfere with your experiments. Approximately

ten thorough rinses are suggested.

Making Your Stock SolutionsFor each solution, find the bottle of the chemical you need and make sure the formula weight on

the label matches your calculations. Weigh out the amount of the chemical you need and write

down the value on the scale read-out. Next, add the material to a beaker that is appropriate forthe volume you intend to make. Place a stir bar in the beaker and add half the total volume of

ddH2

O. Do not add all the water at this time; you need to save room for the acid. Stir thesolution until the entire solid is dissolved. If the solid doesnt fully dissolve after several minutes,add more H2O. When the entire solid is dissolved, pH the solution if needed. Adjust the pH of

your Tris buffer as needed with HCl solution. Then, carefully pour the solution into a graduated

cylinder of appropriate volume and fill to the desired final volume.

Pour the solution into a bottle and label the bottle with the chemical, concentration, pH (if

relevant), names, and date.

Note: Use red tape if youre in Tuesdays lab, blue tape if youre in Wednesdays lab, and

green tape if youre in Thursdays lab.

Repeat this for all of your solutions.

Making the Buffers You Need from Your Stock SolutionsTo test if you made your phosphate buffers properly, you can mix your phosphate buffers for a

final desired pH according to the table in Appendix III. Make 10 mL of 20 mM phosphate bufferat a pH of 6.5 according to the Table and have your TA test the pH with the pH meter or litmus

paper.

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Data to be turned in for Laboratory 2 (25 pts)

1. Make a table like the one below. It should define all six of your buffer solutions and havea place to write down how much material was actually weighed out. Fill in the first fivecolumns before coming to lab, and fill in the last column as you work.

Example: For 50mL of a 0.5 M Cobalt Chloride solution:

Solution Chemical UsedFormula Weight

(g/mol)Volume

Nominal

WeightActual

Weight

0.5 M Cobalt

Chloride

Cobalt Chloride

Hexahydrate237.93 50 mL 5.948 g 5.95 g

2. Tris Buffera. How much HCl solution (and which one) was used to adjust the Tris buffer?b. What was the actual pH of your Tris buffer?

3. Phosphate Buffera. How much of each sodium phosphate solution did you use?b. What was the actual pH of your phosphate buffer?

You should write this in your notebook and turn in the carbon copyyou will lose points if

your TA cannot read and easily understand what you wrote!

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Laboratory 3: PCR Amplification & Gel Electrophoresis

I. Introduction

DNA amplification has become a critical technique for many in the biomedical areas since its

invention in 1983. Using a technique called the polymerase chain reaction (PCR), scientists have

been able to produce a relatively large amount of a targeted piece of DNA from a very smallnumber of starting copies. (Review your lecture notes to re-familiarize yourself with the

process.) The template can be from any form of DNA from a drop of blood, a single hair follicle,or cheek cells, and PCR is used to generate millions of copies of a desired DNA fragment.

It has been estimated that from the 23 pairs of chromosomes (46 total), which comprise human

genomic DNA, a total of 30,000 to 50,000 genes are present. Each gene contains the code for

any given protein. Interestingly, these 30,000 to 50,000 genes comprise only about 5% of the

chromosomal DNA. The remaining 95% is non-coding DNA.

From years of evolution, intron sequences have been targeted by random insertions of short

interspersed elements (SINES). One such repetitive element is known as the Alu sequence. Itsname is derived from a single recognition site for the restriction enzyme AluI. This is a DNA

sequence approximately 300 bp long that is repeated, one copy at a time, almost 500,000 times

within the human genome. The origin and function of this randomly repeated pattern are not yetknown.Alu elements are only in primates, so all of the hundreds of thousands ofAlu copies have

accumulated in primates since their separation from other vertebrates more than 65 million years

ago.

Alu is a transposable DNA sequence that reproduces by copying itself and inserting into new

chromosome locations. It is a retroposon, requiring reverse transcriptase (rt) to make a mobile

copy of itself. Most Alu insertions occur in non-coding regions and are thought to be

evolutionarily neutral. However, an Alu insertion in the NF-1 gene causes neurofibromatosis I,and insertions in introns of genes for the angiotensin converter enzyme (ACE) are associated

with heart disease. Many of these Alu elements have characteristics that make them extremelyuseful to genetic study. In this experiment, you will analyze Alu repeats at a specific

chromosomal location in a number of unknown samples in order to estimate the frequency of this

insert within a population.

This experiment examines PV92, which is a human-specific Alu insertion on chromosome 16.

The PV92 genetic system has only two allelesthe presence or absence of the Alu transposable

element on each of the paired chromosomes. Therefore, there are three PV92 genotypes (++, +-,or --). These two different alleles can be separated by gel electrophoresis. (Review your lecture

notes to re-familiarize yourself with this process.) PV92 is dimorphic, meaning that the elementis present in some individuals and not others. Some have the insert in one copy of chromosome16, and some have it in both copies. The presence of the insert can be detected through PCR

followed by agarose gel electrophoresis.

There are three possible outcomes after your PCR products are electrophoresed. If both

chromosomes have theAlu inserts, each amplified PCR product will be 941 bp long and will

correspond to one band. If there are no inserts on either chromosome, the product will be 641 bp

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long and correspond to one band. (Note: these lengths depend on the primers used.) If there is an

insert on one chromosome but not the other, you will have two different bandsone for the 641bp product and one for the 941 bp product as shown in the example in Figure 1. The bands will

be visualized using ethidium bromide a DNA intercalator. Intercalation causes ethidium bromide

to fluoresce under ultraviolet light (UV). Exposing the gel to UV light after staining allows you

to see bright, pinkish-orange bands where there is DNA.

941 bp PCR product

641 bp PCR product


This handout. Chapter 14 ofFundamental Laboratory Approaches for Biochemistry and Biotechnology

by Ninfa, Ballou, and Benore.

III. Pre-Lab

Design of sequencing primers

The PV92 locus sequence with theAlu insert in bold is:

AACTGGGAAAATTTGAAGAGAAAGTCACACAGATACATTTCAGTAAGGTTGTCTCT

GTTACTTGAGGCTTACAAGAAGGAAAGAAGGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATCACGAGGTCAGGAGATC

GAGACCATCCCGGCTAAAACGCTGAAACCTCGTCTCTACTAAAAATACAAAAAA

TTAGCCGGGCGTAGTGGCGGGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAG

GCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCC

TGCCACTGCACTCCAGCGTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAA

AAAAAAAAAAAAAAAAGAAAGAATTCCCTCTCTAAACACACTCTAACACACAGGAG

TTGAGAACTCA

The primers provided for your PCR reaction are designed further upstream and downstream from

this sequence. Because the primers are propriety, we cannot tell you their exact sequence.

However, you can gain a better understanding of the design process by performing the followingexercise.

Propose a set of primer sequences (approximately 25 bp) that would amplify anyAlu sequence

inserted in the genome. Remember the reverse primer will be the complement of the sequenceshown and that we design primers from the 5 to the 3 direction. Now propose primers for the

PV92 sequence above. What would the PCR product size be with and without the Alu insertion?

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24

How was theAlu sequence named?

Go to the New England Biolabs NEBcutter website:http://tools.neb.com/NEBcutter2/index.php

Paste the PV92Alu sequence into the search box.

Click on Submit to view restriction sites found within theAlu sequence.Does your sequence contain anAlu I restriction site? How many?

IV. Materials

DNA Isolation

100 L saliva 25 L Buffer ATL 10 L Proteinase K

100 L Buffer AL solution (1 Ldissolved RNA carrier stock per 100

L buffer, prepared by your TA)

QIAamp MinElute column 2 mL collection tubes 500 L Buffer AW1

700 L Buffer AW2 700 L ethanol 1.5 mL Eppendorf tubes

15 mL disposable centrifuge tube Water bath (56C) Timer Centrifuge

PCR

20 L complete master mix per PCR sampleo 100 mM Tris buffer (pH ~8.3)o Deoxyribonucleotide triphosphates (dNTPs) - All four bases (dATP, dTTP, dCTP,

and dGTP), 1.6 mM totalo 3 mM MgCl2o 1 M of Forward primero 1 M of Reverse primero 0.05 units/L Taq DNA polymerase*Note that the final concentration in your sample will be half as concentrated.

20 L of each control (+/+, +/-, -/-) 0.2 mL PCR tubes Sharpie marker Ice bath PCR Thermocycler

Agarose Gel Electrophoresis

2g agarose (yields a 1% agarose gel) 1X Tris, Acetate, EDTA Buffer

(TAE)

250 mL Erlenmeyer flask

Hot/stir plate Stir bar 15 L ethidium bromide
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Plastic cassette and comb for gelcasting

5 L loading dye per sample 10 L DNA ladder

100V source UV light

V. Procedure

DNA Isolation

1. While holding the tube with a KimWipe, spit at least 0.5 mL into a disposable 1.5 mLEppendorf tube.

2. Pipette 100 L of your saliva sample into a 1.5 mL Eppendorf tube.3. Add 25 L Buffer ATL and 10 L proteinase K to the sample.4. Pipette in 100 L Buffer AL to the sample.5. Pulse-vortex the sample for about 15-20 seconds.6. Place samples in the 56C water bath for five minutes. Vortex the samples. Put them back in

the water bath for five more minutes.

7. Centrifuge the sample for approximately 30 seconds to remove the drops that formed on theinside of the lid. Note: Be sure to balance the centrifuge when you use it with another

groups sample.

8. Place a QIAamp MinElute column into a 2 mL collection tube, and transfer the lysate(supernatant) to the column. Be sure to dispense the lysate directly onto the silica column,

avoiding the rim and side wall. Do not get the rim wet. Centrifuge this at 8,000 rpm for oneminute. If the lysate has not completely passed through the membrane after this

centrifugation, centrifuge at a higher speed until all liquid has eluted through the column.

9. Put the QIAamp MinElute column in a clean 2 mL collection tube, and discard the collectiontube containing the flow-through. Contact between the column and the flow-through shouldbe avoided.

10.Open the column and add 500 L Buffer AW1 without wetting the rim. Centrifuge again at8,000 rpm for one minute.

11.Put the QIAamp MinElute column in a clean 2 mL collection tube, and discard the collectiontube containing the flow-through.

12.Open the column and add 700 L Buffer AW2 without wetting the rim. Centrifuge at 8,000rpm for another minute.

13.Put the column in a clean 2 mL collection tube and discard the collection tube containing theflow-through.

14.Open the column and add 700 L ethanol without wetting the rim. Centrifuge at 8,000 rpmfor one minute. Put the column in a 2 mL collection tube and discard the collection tube

containing the flow-through.

15.Centrifuge at 14,000 rpm for three minutes to dry the membrane.16.Place the QIAamp MinElute column in a clean 1.5 microcentrifuge tube, and discard the

collection tube containing the flow-through. Open the lid of the QIAamp MinElute column

and incubate at 56C for three minutes.17.Elute the sample by adding 50 L distilled water to the center of the membrane. Your water

should be at room temperature.

18.Incubate at room temperature for three minutes and then centrifuge at 14,000 rpm for oneminute.

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PCR

Note: Be sure to keep all PCR reagents on ice at all times and always wear glovesyour

skin may transfer DNAse to your sample.

1.

Combine 20 L of your sample with 20 L complete Master Mix in a 0.2 mL PCR tube.2. Label your PCR tube with your initials and station number. To prevent the Sharpie fromwiping off, please label on the cylindrical portion of the tube, avoiding the cap and conicalportion.

3. Your TA will prepare three controls. Each control will be prepared in a separate PCR tube bycombining 20 L of the control with 20 L complete master mix. Mix well by flicking the

PCR tube.4. Give your samples (still on ice) to your TA to run the PCR. The PCR takes about 1.5 hours.

While you wait, prepare your 1% agarose gel.

5. The thermal cycling parameters are as follows.Note: The denature, anneal and extend times have been reduced to 30 seconds each.

Gel Electrophoresis1. You will be sharing gels, and your TA will tell you who will be sharing with whom. Each gel

takes about 1.5-2.0 L 1x TAE, so plan your procedure accordingly.

2. Each gel takes one cassette. Tape the ends of your cassette and place the comb towards thetop. Your TA will demonstrate how it should look.

3. Prepare 1.5 L of 1x TAE. You will need to dilute the stock solution.4. In a 250 mL E. flask, add 1.5 g agarose to 150 mL 1x TAE.5. Place a stir bar in the flask and heat it at medium-high heat while stirring. You will know it is

finished heating when all the agarose is dissolved and large bubbles cover the bottom of your

flask.6. Remove from heat and remove the stir bar using a larger stir bar.7. Add 12 L ethidium bromide. Try to get all the bromide out of the pipette tip by pipetting up

and down in the agarose solution. Be very careful when using the ethidium bromide and be

sure to cap it as soon as you get your portionit is a DNA intercalator!

8. Swirl your flask to uniformly distribute the ethidium bromide. Allow it to cool for a fewminutes.

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9. Slowly pour your solution into the cassette until your gel is about 1 cm deep. Be sure thatyour comb is not completely submerged.

10.Let the gel cool and harden. It should be ready when your PCR samples are ready.11.Once the PCR is complete, remove the comb and put the cassette into the gel chamber. Fill

the chamber with 1x TAE.

12.Add 5 L of loading dye into each of your PCR tubes and mix well by pipetting up anddown.

13.The TA will load 10 L of the DNA ladder into the first well in the gel to demonstrate howto load a gel. The DNA and loading dye solution is heavier than water, so it will sink to the

bottom of the well. Dispense it gently and try not to agitate the buffer in the vicinity of the

well. When loading, do not completely empty your pipette. The last push may contain an air

bubble that will disturb your sample.14.The TA or some of the students will load 35 L of each of the controls.15.You will each load 35 L of your own sample into separate wells. Keep track of which well

contains what samplewrite it down. Prepare a table of what is in each lane.16.Place the top on the chamber and plug it in. The black wire is negative, and the red wire is

positive. Based on your knowledge of electrophoresis, figure out which wire should beplugged in to what side.17.Turn on your 100V source and let the gel run. Look for little bubbles forming where the

black wire is connected to be sure that the gel is receiving the power. The electrophoresis

should take about an hour.18.Turn off the power supply and carefully remove the gel from the chamber.19.Use the UV lamp to visualize your results. Be sure to wear goggles. Do not look into the UV

light directly.

20.Tabulate the results of each lane (e.g., ++, +-, --, no bands) in your notebook. Report yourresults to your TA when you finish. The TA will e-mail images of all the gels from each

section to the class list so you can complete your statistical analyses for your report.

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Guidelines for the PCR Amplification & Gel Electrophoresis Lab Report

Before you get started, be sure to survey the literature regarding your results.

Prepare your figures first. You must prepare any figures yourselfdo not take figures from

websites or articles. Create a figure for your DNA agarose gel and clearly identify the allelesdetected in each sample case. Use software such as Microsoft Word to crop the image and label

the lanes. The figure should look professional. Figures should be numbered in the order in whichthey appear, and have titles and figure legends.

Gather your references. Beyond textbook knowledge, you should be referencing primaryliterature. Wikipedia and web sites should only serve as a starting point; they should not be used

in your reference section. You may want to learn about referencing software such as Endnote,

which makes referencing a long document easier.

Be sure to proofread the entire report. Points will be deducted for poor grammar, spelling

mistakes, awkward wording, and lack of clarity.

General formatting: Margins: 1 all sides Font size: 11 point Arial 1.5 line spacing Figures should be numbered and attached to the back of the report Figure legends: 10 point and single spaced Section headings should not be cut off to another page

Title Page

The title page should only include the following: Title of the lab Your name (dont include your name on any other page of the report) The name(s) of your partner(s) A signed honor code pledge. The date turned in

Abstract (7 pts) 0.5 page

Your abstract should be a thorough but concise synopsis of your report. It should summarize the

results of the experiments and state any significant conclusions. The abstract should be no morethan five sentences (typically between 50-100 words). Usually, the abstract is written last. You

want to include the goal of the experiments, a summary of the results, and a concludingstatement about the results. The abstract can be very difficult to write, so stick to the big picture

and report the results and conclusion. There is no room for details or background information.

Introduction (12 pts) 1.5 pagesThe introduction should state the purpose of the experiment and give a brief outline of the

necessary theory, which is often done by citing pertinent primary literature. You can include a

very short description of the methods used. This section should present a clear statement of the

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aims of the experiment and/or the hypothesis being tested. Assume the reader has a background

in general chemistry and biochemistry. Do some research on the Alu sequence and be sure toexplain the significance to this lab report using additional citations from primary literature. Use

the present tense throughout this section.

Materials and Methods (12 pts) 1 page or lessDo not include specific volumes and every detail. Do not copy the protocol from your lab write-

ups. Do not use lists or tables of reagents. Many of the methods you have used are standardprotocols. You want to write in sentence form briefly describing what you did and with what

reagents. This section should be written in the past tense and follow a chronological order.

Results (16 pts) 0.5 page, not including figures and tablesJust report the results. Use the text to explain your results and refer to the figure that represents

these results. Do not elaborate further than a description of the result. Raw data should not be

included. Your figures should be polished and helpful to the reader with regions labeled, arrowsindicating points of interest, and complete legends. Each figure and legend should be on a

separate page and assembled in order after the reference section. Each figure should benumbered and have a descriptive title. Be sure to refer to the figures and tables in the text.

Calculate the genotype distributions for the entire class population (your TA will email you all

the gels and indicate which lanes are the ladder and controls) and the specific allele frequencies.An allele frequency is a ratio comparing the number of copies of a particular allele to the total

number of alleles present. Here is an illustration:

Imagine a class of 100 students with the following genotype distribution:+/+ 20 +/- 50 -/- 30

Since humans are diploid, the total number of alleles in the class is:2 x 100 = 200.

The allele frequency for PV92+ is:

2 x 20 (homozygotes) + 50 (heterozygotes) / 200 = 90 / 200 = 0.45Likewise, the allele frequency for PV92- is:

2 x 30 (homozygous) + 50 (heterozygotes) / 200 = 110 / 200 = 0.55

If you are including any sample data provided by the TAs, you need to include your data along

with the sample data. All of your results should be includeddo not exclude results just because

they are unexpected or inconsistent with other data. Discrepancies should be pointed out andexplored in the discussion section.

Discussion (18 pts) 1 page, not including figures

Do not introduce data in this section. Briefly explain the experimental goals and if they wereattained. Follow with a more detailed interpretation of your data. Do you feel your results are

reliable? Why or why not? Do not attribute experimental failure to the equipment or procedures

in the write-up. (They really do work as seen in the sample data.) Also, do not use sweepingvague statements as human error to describe discrepancies in expected versus observed data.

Be specific and explain possibilities that could have contributed to the observed results.

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How do your results compare to those reported in the literature? Be sure to reference the data

you compare your results to. Which values did you chose to compare to your values and why? Ifyour values are not in agreement with the reported values, why do you think this is the case?

Comment on the specific frequency of each allele in your overall class sample.

Using the genotype distribution from your class, calculate the frequencies of the + and - allelesof PV92.If you need help with this, see your TA. You should be able to do a literature searchand the use Allele Serverhttp://www.bioservers.org/html/sad/sad.html to find the expectedfrequencies and distributions in other populations, which will allow you to compare them to the

class data. (Log-on as guest, go to manage groups, and select reference groups.)

Based on the results you recorded compared to the reported data, how useful is the PV-92Alu

polymorphism in distinguishing populations from each other? Why do you think that the PV-92

allele frequencies differ significantly between some populations, while not between others? Do

you think you could use PV-92 data to answer the questions of where humans originated and thepaths by which they spread throughout the world?

Total page length (not including references, figures and tables, and questions) should not exceed4.5 pages.

Questions (10 pts)Include the answers to the following questions at the end of the lab. You may turn in handwritten

answers if you choose, but they must be written in pen and on white, unlined paper. Not doing so

will result in a deduction of 5 points.

1. What is needed from the cells for PCR? What structures must be broken to release DNAfrom a cell?

2. What is the purpose of the complete master mix? Using what you know about PCR, whatare its components and their respective roles?

3. Why is it necessary to have a primer on each side of the DNA segment to be amplified?4. Describe the three main steps of each cycle of PCR amplification and what reactions occur

at each temperature.

5. Explain why the precise length target DNA sequence does not get amplified until the thirdcycle.

6. What is the difference between an intron and exon?7. Why do the possible PCR products differ in size by 300 base pairs?8. What determines how far DNA fragments migrate on the gel? Why is a voltage applied to

the gel?

9. Why is the PCR run before the gel electrophoresis?10.What is the purpose of the DNA ladder?11.What controls did you run in this experiment? Why are they important?12.Propose an explanation of the third band (greater than 1 kb) in the heterozygote samples.

ReferencesThe references should be on a separate page at the end of the report, before the figures and

tables. Please use a superscripted number in the text that corresponds with the number in the

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reference list. These five points are solely for formattingthey do not correspond to the number

of primary citations, which is part of your discussion section grade. Use the following examplesas a format for your citations (include the title of the article) in the reference section.

Journal articles:

1. Mallick, P., Boutz, D.R., Eisenberg, D., and Yeates, T.O. 2002. Genomic evidence that theintracellular proteins of archaeal microbes contain disulfide bonds. Proc. Natl. Acad. Sci. 99:96799684.

Book chapters and sections:

2. Yu, Y.-T., Scharl, E.C., Smith, C.M., and Steitz, J.A. 1999. The growing world of small

nuclear ribonucleoproteins. In The RNA world, 2nd ed. (eds. R.F. Gesteland et al.), pp. 487524.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

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Laboratory 4: Quantitative Determination of Protein Concentration

I. Introduction

Protein quantitation is often necessary before processing protein samples for isolation,separation, and analysis by chromatographic, electrophoretic and immunochemical methods. The

most common methods for the colorimetric detection and quantitation of total protein can bedivided into two groups based upon the chemistry involved. Protein assay reagents involve either

protein-dye binding (e.g., coomassie) chemistry or protein-copper chelation chemistry. When itis necessary to determine total protein concentration in a sample, one must first select an

appropriate protein assay method. The choice among available protein assays usually is based

upon the compatibility of the method with the samples to be assayed. The objective is to select amethod that requires the least manipulation or pre-treatment of the samples containing

substances that may interfere with the assay. Each method has its advantages and disadvantages

(see Table 3.3 on page 71 of your textbook). Because no one reagent can be considered to be theideal or best protein assay method for all circumstances, most researchers have more than one

type of protein assay reagent available in their lab.

Selection of a Protein Standard

Selection of a protein standard is potentially the greatest source of error in any protein assay. Of

course, the best choice for a standard is a purified, known concentration of the predominant

protein found in the samples. This is not always possible or necessary; in some cases, all that isneeded is a rough estimate of the total protein concentration in the sample. For example, in the

early stages of purifying a protein, identifying which fractions contain the most protein may be

all that is required. If a highly purified version of the protein of interest is not available or it istoo expensive to use as the standard, the alternative is to choose a protein that will produce a

very similar color response curve with the selected protein assay method (see following section

on Protein-Protein Variation). For general protein assay work, bovine serum albumin (BSA)

works well for a protein standard because it is widely available in high purity and relativelyinexpensive. Although it is a mixture containing several immunoglobulins, bovine gamma

globulin (BGG) also is a good standard when determining the concentration of antibodies, since

BGG produces a color response curve that is very similar to that of immunoglobulin G (IgG).

For greatest accuracy in estimating total protein concentration in unknown samples, it is essential

to include a standard curve each time the assay is performed. This is particularly true for theprotein assay methods that produce non-linear standard curves. Deciding on the number of

standards and replicates used to define the standard curve depends upon the degree of non-

linearity in the standard curve and the degree of accuracy required. In general, fewer points areneeded to construct a standard curve if the color response is linear. Typically, standard curves are

constructed using at least two replicates for each point on the curve.

Sample Preparation

Before a sample is analyzed for total protein content, it must be solubilized, usually in a buffered

aqueous solution. Additional precautions are often taken to inhibit microbial growth or to avoidcasual contamination of the sample by foreign debris such as dust, hair, skin or body oils.

When working with tissues, cells, or solids, the first step of the solubilization process is usually

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disruption of the samples cellular structure by grinding and/or sonication or cell lysis by the use

of specially designed reagents containing surfactants to lyse the cells. This is done in aqueousbuffer containing one or more surfactants to aid the solubilization of the membrane-bound

proteins, biocides (antimicrobial agents) and protease inhibitors. After filtration or centrifugation

to remove the cellular debris, additional steps such as sterile filtration, removal of lipids or

further purification of the protein of interest from the other sample components may benecessary.

Non-protein substances in the sample that are expected to interfere in the chosen protein assay

method may be removed by dialysis, gel filtration, or precipitation.

Protein-to-Protein Variation

Each protein in a sample responds uniquely in a given protein assay. Such protein-to-protein

variation refers to differences in the amount of color (absorbance) obtained when the same mass

of various proteins is assayed concurrently by the same method. These differences in colorresponse relate to differences in amino acid sequence, isoelectric point (pI), secondary structure,

and the presence of certain side chains or prosthetic groups.

Protein-to-protein variation may be a consideration in selecting a protein assay method,

especially if the relative color response ratio of the protein in the samples is unknown. As

expected, protein assay methods that share the same basic chemistry show similar protein-to-protein variation. These data make it obvious why the largest source of error for protein assays is

the choice of protein for the standard curve.

Calculation of Results

When calculating protein concentrations manually, it is best to use point-to-point interpolation.

This is especially important if the standard curve is non-linear. Point-to-point interpolation refersto a method of calculating the results for each sample using the equation for a linear regression

line obtained from just two points on the standard curve. The first point is the standard that has

an absorbance just below that of the sample and the second point is the standard that has anabsorbance just above that of the sample. In this way, the concentration of each sample is

calculated from the most appropriate section of the whole standard curve. Determine the average

total protein concentration for each sample from the average of its replicates. If multipledilutions of each sample have been assayed, average the results for the dilutions that fall within

the most linear portion of the working range.

When analyzing results with a computer, use a quadratic curve fit for the non-linear standard

curve to calculate the protein concentration of the samples. If the standard curve is linear, or if

the absorbance readings for your samples fall within the linear portion of the standard curve, the

total protein concentrations of the samples can be estimated using the linear regression equation.

Most software programs allow you to construct and print a graph of the standard curve, calculate

the protein concentration for each sample and display statistics for the replicates. Typically, thestatistics displayed will include the mean absorbance readings (or the average of the calculated

protein concentrations), the standard deviation (SD) and the coefficient of variation (CV) for

each standard or sample. If multiple dilutions of each sample have been assayed, average the

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results for the dilutions that fall in the most linear portion of the working range.

For this experiment you will determine the protein concentration of three unknowns using a

calibration curve from a standard solution of bovine serum albumin (BSA). The unknown protein

concentrations will be between 0.5 mg/mL and 2.0 mg/mL.

You will be given two unknown protein solutions at concentrations between 0.5 and 2.0 mg/mL.

For each unknown you are to determine the protein concentration by the Lowry, BCA andBioRad methods. To do this you will start by constructing a standard curve (absorbance vs.

amount of protein) using a standard solution of BSA (1.0 mg/mL) for each assay.

Tips/Notes

1. Plan out (with your lab partner) how you can efficiently make and measure each proteinsample so that you give each sample approximately the same incubation time. It would be

wise to measure out the buffer and the assay reagent first and add the protein last.2. Please note that each assay requires different wavelengths for absorbance

measurements. Remember to change them accordingly!3. Also note: CuSO4 5H2O used in the Lowry reagent and the Copper (II) SulfatePentahydrate solution used in the BCA assay are different!

4. Be sure to mix your solutions well. The best way is to invert the cuvette or tube six times.5. Be sure to use the right volume cuvette for the volume you are using. For example, do not

put 1 mL into a 4 mL cuvette.

6. Be sure that you understand the spectrophotometer that you are using (some are designeddifferently). Where is the light source coming? Where is the detector? How full should

your cuvette be so that your sample is aligned with the light source?


This handout Chapters 3 & 4 of Fundamental Laboratory Approaches for Biochemistry andBiotechnologyby Ninfa, Ballou, and Benore.

III. Pre-Lab

1. Calculate how much stock BSA (10 mg/mL) is needed to make the amount of BSA neededfor this lab (1.0 mg/mL). (Remember that BSA is needed for all three assays and everything

needs to be done in duplicates.)_____________ mL of stock BSA to make _____________ mL of dilute BSA

2. Calculate how many grams of each reagent you need to make the Lowry reagent:a. NaOH (s): ____________ g in ____________ mLb. Sodium carbonate: ____________ g in ____________ mLc. Sodium tartrate*: ____________ g in ____________ mLd. CuSO4.5H2O*: ____________ g in ____________ mL

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35

Note: It would be very difficult to make 500 L of these solutions and still maintain the

correct % w/v. It is suggested that you make a bit more than what you need for better

accuracy. Discuss this with your lab partner prior to lab.

3. Fill out the protein concentration columns for each assays standard curve table.4. Calculate the range of unknown protein volumes needed to ensure the concentration is withinthe upper and lower boundaries of the standards5. Calculate two sample volumes within the range you found in step 2 and put your values in

the table given below. Be sure to cover the whole range (present all values in L).

Assay Range Tube 1 (x) Tube 2 (y)

Lowry

BCA

BioRad

Sample Calculation:How to calculate the amount of the unknown protein solution to use:Lets say your BSA standard concentration is 2 mg/mL and the unknown protein solution

concentration ranges from 0.1 mg/mL to 0.2 mg/mL.

Example standard table:

Tube Number

Assay Reagent + Buffer

(L)

Protein

Standard

(L)

Protein

Concentration(mg/mL)

1 1995 5 0.005

~ ~ ~ ~

6 1990 10 0.010

Recall: Concentration1 Volume1 = Concentration2 Volume2.

1. In the case that the unknown protein concentration is 0.1 mg/mL:a. (0.005 mg/mL)(2 mL) = (0.1 mg/mL)(?) ? = 0.1 mLb. (0.010 mg/mL)(2 mL) = (0.1 mg/mL)(?) ? = 0.2 mL

2. In the case that the unknown protein concentration is 0.2 mg/mL:a. (0.005 mg/mL)(2 mL) = (0.2 mg/mL)(?) ? = 0.05 mLb. (0.010 mg/mL)(2 mL) = (0.2 mg/mL)(?) ? = 0.1 mL

The range in this case is from 50L to 200L.

IV. Materials

Vortex Micropipettes (P20, P200,

P1000)

Distilled H2O Spectrophotometer

125 mL Erlenmeyer flask 24 - 13 x 100 mm test tubes Test tube rack 6 - 4 mL cuvettes (for the Lowry

Assay)


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12 - 1.5 mL cuvettes (6 for BCAand 6 for BioRad)

Sharpie marker 48 - 1.5 mL Eppendorf tubes Eppendorf tube rack Graduated cylinder Timer 2 - 50 mL screw-top plastic

tubes

1 - 10 mL screw-top plastictubes

Scoopula Weigh paper Balance Water bath set at 37C 10 mL of 10 mM phosphate

buffer (pH 7.0) (this is referredto as buffer throughout)

V. Procedures

Part A. The Lowry Assay1.Prepare the Lowry reagent:

Combine the reagents listed below in a 125 mL Erlenmeyer flask, swirling after each

addition.

Note: It is very important to add the reagents in the order written.

Volume Components (all % are weight/volume)50 mL 2% sodium carbonate in 0.1 N NaOH

500 L 1% sodium tartrate

500 L 0.5% CuSO4 5H2O

2.Lowry Assay:a. Use disposable 13 x 100 mm test tubes. Set up all tubes at once so that they incubate for

approximately the same amount of time. Be sure to use a clear labeling system to avoidtube mix-ups.

b. Set up standard curve and unknown solutions according to the table below.DoNOTadd the Folin and Ciocalteu's Phenol at this point.

c. Vortex each tube and wait at least 10 minutes. Make sure that your solution does notspill over. Use your timer.

d. Add 200 L of Folin and Ciocalteu's Phenol reagent and vortex thoroughly. (Thissolution will be made fresh daily by the TA.)

e. Wait 20 minutes. Set your timer so you can move on to set up the next part of yourexperiment while you wait. (Note: The final volume of this assay is 2.4 mL, not 2.2 mL.)

3.Using 4 mLplastic cuvettes, record the absorbance at 750 nm.

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STANDARD CURVE: This must be done in duplicate.

Standard Curve

TubeLowry

Reagent (uL)Buffer (uL)

Vol Standard

(uL)

Folin Reagent

(uL)

Conc.

(mg/mL)

Absorbance

1 2

1 2000 200 0 200

2 2000 195 5 200

3 2000 190 10 200

4 2000 185 15 200

5 2000 180 20 200

6 2000 175 25 200

UNKNOWN SAMPLES: Select values ofx and y so that their absorbance values fall within

the standard curve. Both solutions must be done in triplicate foreach unknown.

Unknown A

Lowry Reagent

(uL)

Buffer

(uL)

Folin Reagent

(uL)

Vol Added

(uL)

Absorbance

1 2

Replicate

3

A1 2000 200-x 200 x

A2 2000 200-y 200 y

Unknown B

Lowry Reagent

(uL)

Buffer

(uL)

Folin Reagent

(uL)

Vol Added

(uL)

Absorbance

Replicate

1

Replicate

2

Replicate

3

B1 2000 200-x 200 x

B2 2000 200-y 200 y

Note: All absorbencies and concentrations should be recorded in your lab notebook. These

tables are only for you to use as a formatting reference.

Part B. The BCA Assay1. Prepare the BCA reagent:

Mix 25 mL of Bicinchoninic Acid Solution (Sigma) with 0.5 mL of Copper (II) Sulfate

Pentahydrate in a 50 mL plastic screw-top tube. Vortex this to mix well.

2. BCA Assay:a. Use 1.5 mL microcentrifuge tubes for these samples. Set up all tubes at once so that theyincubate for approximately the same amount of time. Be sure to use a clear labelingsystem to avoid tube mix-ups.

a. Use the same BSA standard as was used in the previous method. Set up standard curveand unknown solutions according to the table below.

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a. Vortex each sample and place them in the foam rack. Submerge these in a 37C waterbath for at least 10 minutes.

3. Using 1.5 mL plastic cuvettes, record the absorbance at 562 nm.

STANDARD CURVE: This must be done in duplicate.Standard Curve

TubeBCA Reagent

(uL)Buffer (uL)

Vol Standard

(uL)

Conc.

(mg/mL)

Absorbance

1 2

1 1000 50 0

2 1000 47 3

3 1000 44 6

4 1000 40 10

5 1000 35 15

6 1000 30 20

UNKNOWN SAMPLES: Select values ofx and y so that their absorbance values fall withinthe standard curve. Both solutions must be done in triplicate foreach unknown.

Unknown A

BCA Reagent (uL)Buffer

(uL)

Vol Added

(uL)

Absorbance

1 2

Replicate

3

Ax 1000 50-x x

Ay 1000 50-y y

Unknown B

BCA Reagent (uL) Buffer(uL)

Vol Added(uL)

AbsorbanceReplicate

1Replicate

2Replicate

3

Bx 1000 50-x x

By 1000 50-y y



Part C. The BioRad Assay1. Prepare the BioRad reagent:

You will need approximately 25 mL diluted BioRad dye. The dilution of the dye shouldbe 4 parts water to 1 part dye. Combine in a 50 mL screw-top plastic tube. Vortex the

solution to mix it well.

2. BioRad Assay:a. Use 1.5 mL microcentrifuge tubes for these samples. Set up all tubes at once so that

they incubate for approximately the same amount of time. Be sure to use a clearlabeling system to avoid tube mix-ups.

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b. Use the same BSA standard as was used in the previous methods. Set up standard curveand unknown solutions according to the table below.

c. Briefly vortex each tube.3. Record the absorbance in 1.5 mL plastic cuvettes at 595nm.

STANDARD CURVE: This must be done in duplicate.

Standard Curve

TubeBioRad Dye

(uL)Buffer (uL)

Vol Standard

(uL)

Conc.

(mg/mL)

Absorbance

1 2

1 1000 20 0

2 1000 18 2

3 1000 16 4

4 1000 14 6

5 1000 12 86 1000 10 10

UNKNOWN SAMPLES: Select values ofx and y so that their absorbance values fall within

the standard curve. Both solutions must be done in triplicate foreach unknown.

Unknown A

BioRad Dye (uL)Buffer(uL)

Vol Added(uL)

Absorbance

1 2

Replicate

3

Ax 1000 20-x x

Ay 1000 20-y y

Unknown B

BioRad Dye (uL)Buffer(uL)

Vol Added(uL)

Absorbance

Replicate1

Replicate2

Replicate3

Bx 1000 20-x x

By 1000 20-y y



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Guidelines for Quantitative Determination of

Protein Concentrations Lab Report (100 pts)

Before you get started, be sure to survey the literature regarding your results.

Prepare your figures first. You must prepare any figures yourselfdo not take figures fromwebsites or articles. Use software such as Microsoft Word to crop the image and label the lanes.

The figure should look professional. Figures should be numbered in the order in which theyappear, and have titles and figure legends.

Gather your references. Beyond textbook knowledge, you should be referencing primary

literature. Wikipedia and web sites should only serve as a starting point; they should not be used

in your reference section. You may want to learn about referencing software such as Endnote,

which makes referencing a long document easier.

Be sure to proofread the entire report. Points will be deducted for poor grammar, spelling

mistakes, awkward wording, and lack of clarity.

General formatting: Margins: 1 all sides Font Size: 11 point Arial 1.5 line spacing Figures should be numbered and attached to the back of the report Figure legends: 10 point and single spaced Section headings should not be cut off to another page.

Title Page

The title page should only include the following. Title of the lab Your name (dont include your name on any other page of the report) The name(s) of your partner(s) A signed honor code pledge. The date turned in

Abstract (10 pts) 0.5 page

Your abstract should be a thorough but concise synopsis of your report. It should summarize the

results of the experiments and state any significant conclusions. The abstract should be no morethan five sentences (typically between 50-100 words). Usually, the abstract is written last. You

want to include the goal of the experiments, a summary of the results, and a concludingstatement about the results. The abstract can be very difficult to write, so stick to the big pictureand report the results and conclusion. There is no room for details or background information.

Introduction (20 pts) 0.75 pageThe introduction should state the purpose of the experiment and give a brief outline of the

necessary theory, which is often done by citing pertinent primary literature. You can include a

very short description of the methods used. Provide some specifics (e.g., chromophores,

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wavelengths) about each assay, and highlight the differences between the assays. This section

should present a clear statement of the aims of the experiment and/or the hypothesis being tested.Assume the reader has a background in general chemistry and biochemistry. Use the present

tense throughout this section.

Results (35 pts) 1.5 pages, not including figuresJust report the results. Use the text to explain your results and refer to the figure that represents

these results. Do not elaborate further than a description of the result. Raw data should not beincluded. Your figures should be polished and helpful to the reader with regions labeled, arrows

indicating points of interest, and complete legends. Each figure and legend should be on a

separate page and assembled in order after the reference section. Each figure should be

numbered and have a descriptive title. Be sure to refer to the figure in the text.

For each assay, you should have a standard curve plot including both sets of data (do not

calculate the standard deviation) and report the concentrations of the unknown samples.(Remember to take into account any dilutions and report the concentration of the unknown

sample that was given to you before your diluted it.) Include the unknown designation (A,B,C,Dor E), the mean absorbance value and the standard deviation (be sure to throw out data that

does not fall within the standard curve), and the mean calculated protein concentration of the

original unknown solution and the standard deviation.

Make sure you are reporting concentration units and be consistent with the units you are using.

For one set, show a sample calculation for the concentration and the complete calculationfor the standard deviation. For subsequent sets, the calculation may be done by your

spreadsheet program. For each unknown, calculate the accuracy or % error.

Dont report all the steps to get the results, just report the results. Do not include conclusionsthat is, do not analyze and compare the results. Save that for the discussion section.

If you are including any sample data provided by the TAs, you need to include your data alongwith the sample data. All of your results should be includeddo not exclude results just because

they are unexpected or inconsistent with other data. Discrepancies should be pointed out and

explored in the discussion section.

Discussion (30 pts) 1 page, not including figures

Do not introduce data in this section. Briefly explain the experimental goals and if they wereattained. Follow with a more detailed interpretation of your data using this information. Do you

feel your results are reliable? Why or why not? Do not attribute experimental failure to the

equipment or procedures in the write-up. (They really do work as seen in the sample data.) Also,

do not use sweeping vague statements as human error to describe discrepancies in expectedversus observed data. Be specific and explain possibilities that could have contributed to the

observed results. How do your results compare to those reported in the literature with respect to

the sensitivity of the assays?

Compare the unknown concentrations determined with each assay. Which was more accurate?

Suggest reasons why. Your TA will supply you with the actual concentrations of the unknowns

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at the end of the week. What is the difference between accuracy and precision? What was the %

accuracy of your measurements? What was the precision of your measurements?

Do some of your own research on the different methods and why one assay type may be more

suited to your unknown based on your results. Be sure to reference primary literature.

Conclusion (5 pts) 0.25 pageThis section should include the overall conclusions of the labdo not introduce new data, anddo not just summarize your results. State a conclusion based on the data.

Total page length (not including references, figures, and tables) should not exceed four pages.

ReferencesThe references should be on a separate page at the end of the report, before the figures. Please

use a superscripted number in the text that corresponds with the number in the reference list.These five points are solely for formattingthey do not correspond to the number of primary

citations, which is part of your discussion section grade. Use the following examples as a formatfor your citations (include the title of the article) in the reference section.

Journal articles:

1. Mallick, P., Boutz, D.R., Eisenberg, D., and Yeates, T.O. 2002. Genomic evidence that theintracellular proteins of archaeal microbes contain disulfide bonds. Proc. Natl. Acad. Sci. 99:96799684.

Book chapters and sections:2. Yu, Y.-T., Scharl, E.C., Smith, C.M., and Steitz, J.A. 1999. The growing world of small

nuclear ribonucleoproteins. In The RNA world, 2nd ed. (eds. R.F. Gesteland et al.), pp. 487524.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

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Laboratory 5: General Cloning Methods and

Recombinant Protein Expression I

I. Introduction

As part of a protein structure initiative, the following gene was cloned from Pyrobaculumaerophilum:

ATGGCCTCGGATATATCTAAATGCTTTGCCACACTTGGCGCAACATTACAGGACTCGATAGGTAAGCAGGTACTCGTAAAGCTGAGAGATAGCCAC

GAAATAAGGGGGATTTTGCGCTCCTTTGACCAACACGTCAACTTATTG

CTAGAAGATGCAGAAGAAATAATTGACGGAAATGTGTACAAAAGGGGC

ACTATGGTAGTGAGAGGAGAGAACGTACTCTTTATTTCACCAGTACCA

You will to produce and purify this protein. The gene has already been cloned into a

pETT22b(+) vector between the NdeI and HindIII endonuclease restriction sites. The next set oflabs (5 9) is designed so that you can produce and purify the protein product of this gene.

TransformationThere are two methods to transform Escherichia

coli cells with plasmid DNA: chemical

transformation and electroporation. For chemical

transformation, cells are grown to mid-log phase,harvested and treated with divalent cation salts

such as CaCl2. Cells treated in such a way are said

to be competent. To chemically transform cells,competent cells are placed on ice and mixed with

the DNA, exposed to a brief heat shock at 42 C,

and returned to ice. Then, cells are incubated withrich medium and allowed to express the antibiotic

resistant gene for 30-60 minutes prior to plating.

For electroporation, cells are also grown to mid-

log phase but are then washed extensively withwater to eliminate all salts. Usually, glycerol is

added to the water to a final concentration of 10%

so that the cells can be stored frozen and saved forfuture experiments. To electroporate DNA into

cells, washedE. coli are mixed with the DNA to be

transformed and then pipetted into a plastic cuvette

containing electrodes. A short electric pulse, about2400 volts/cm, is applied to the cells causing

smalls holes in the membrane through which the DNA enters. The cells are then incubated withbroth as above before plating. For chemical transformation, there is no need to pre-treat the

DNA. For electroporation, the DNA must be free of all salts so the ligations are first precipitated

with alcohol before they are used.

Figure 1: Cloning gene of interest into

an expression vector.

By PCR Amplification

and restriction digests

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Figure 2: Vector map of the E. coli expression vector pET-22b


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This handout Chapter 13 ofFundamental Laboratory Approaches for Biochemistry and Biotechnologyby

Ninfa, Ballou, and Benore.

III. Pre-labYou should determine the following about your gene of interest and the protein product (use the

suggested websites in Appendix I):

1. Are there any rare codons present in the gene?2. Looking at the vector map on the previous page, is there any modification to the

expressed protein when cloned into the NdeI/HindIII restriction sites?

3. What is the amino acid sequence of the gene product?4. What is the predicted molecular weight of the protein?5. What is the predicted isoelectric point (pI) of the protein?

IV. Materials

Incubator set at 37C (for plates) 250 mL culture (Erlenmeyer) flask Aluminum foil

Ice Water bath set at 42C Bunsen burner and striker

Autoclave tape Shaker/incubator set at 37C (for cultures) Culture tube with vented cap LB (Luria-Bertani)-AMP (Ampicillin) agar

plates P20, P200, and P1000 pipettes Cell sp

Laboratory Manual for Chem4411 Final 072109

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