Faster, Better Biotech: Mini-Preps

Ellyn Daughertyellyn@BiotechEd.comwww.BiotechEd.com

Simon HoldawayThe Loomis Chaffee Schoolsimon.holdaway@gmail.com

The rAmylase Project Model of rDNA/protein Business

< Chapters 1-5 Basic SLOP

< Lab 5f Amylase PAGE (revised)

< Lab 6e Amylase Producing Bacteria

< Lab 6c Amylase Activity Assay (revised)

< Lab 6d Amylase ELISA and W Blot

< Labs 7a/7b/7f/7g Amy Spectrophotometry

< Lab 8b Restriction Digestion

< Lab 4j/8b/8g DNA Gel Electrophoresis

< Lab 8c pAmy Transformation of E. coli

< Lab 9c/9d Amy Ion-Ex Chromatography

< Lab 8g Plasmid DNA Isolation

< Lab 13i Amylase Gene PCR

The goal of The rAmylase Project is to

• Learn how to assay for a protein of commercial interest (amylase)

• Transform cells to produce that protein (using pAmylase and C2523 cells)

• Scale-up cells to volumes where amylase can be purified and assayed

Find a product of interest (amylase) and make sure to have assays to show it’s presence,

activity, & concentration

Amylase is an enzyme that catalyzes starch digestion.

It is used commercially in several ways including:

1)Remove starch in products2)Produce sugar from starch3)Processing of cellulostic biofuel

The rAmylase Project Model of rDNA/protein Business

< Chapters 1-5 Basic SLOP

< Lab 5f Amylase PAGE (revised)

< Lab 6e Amylase Producing Bacteria

< Lab 6c Amylase Activity Assay (revised)

< Lab 6d Amylase ELISA and W Blot

< Labs 7a/7b/7f/7g Amy Spectrophotometry

< Lab 8b Restriction Digestion

< Lab 4j/8b/8g DNA Gel Electrophoresis

< Lab 8c pAmy Transformation of E. coli

< Lab 9c/9d Amy Ion-Ex Chromatography

< Lab 8g Plasmid DNA Isolation

< Lab 13i Amylase Gene PCR

Faster, Better Plasmid Isolation

Lab 8g Zippy TM Plasmid Miniprep Kit (Zymo Research Corp #D4036)

It is often necessary to extract the transforming plasmid from the transformed cells. This is called a preparation (called “prep,” for short).

When the amount of cells and cell culture is small, the amount of plasmid recovered from the cells is relatively small.

A miniprep is a plasmid isolation that yields about 20 to 30 μg of DNA (usually 20 μg in a 50 μL sample).

The “old mini-prep way” (Alkaline lysis) requires cell pelleting and lots of nasty buffers.

But now we have…

Zippy TM Plasmid Miniprep Kit

Faster, Better Plasmid Isolation

Zippy TM Plasmid Miniprep Kit Pre-lab prep

Grow 5 mL of overnight transformed cell culture.

In six 50-mL tubes with 12 mL sterile LB Broth, add 12µL of 1000X amp and 100 µL of overnight culture. Grow 18 hours at 37°C

Use 60 µL of this culture in each mini-prep

Amylase producing DH5-alpha E. coli cells

Faster, Better Plasmid Isolation

Zippy TM Plasmid Miniprep Kit

The fastest, easiest miniprep available for purifying transfection quality plasmid DNA.

Pellet-free procedure omits conventional cell pelleting and re-suspension steps.

DNA quality appropriate for cloning, sequencing, and transfection.

Zyppy Protocol: The following procedure is performed at room temperature.

1.Add 600 µL of bacterial culture grown in LB medium to a 1.5 mL microcentrifuge tube.

2. Add 100 µL of 7X Lysis Buffer (Blue) and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes.After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.

3. Add 350 μL of cold Neutralization Buffer (Yellow) and mix thoroughly.The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization.

4. Centrifuge at 11,000 – 16,000 xg for 2-4 minutes.5. Transfer the supernatant (~900 μL) into the provded Zymo-Spin™ IIN column. Avoid disturbing the cell debris pellet.6. Place the column into a Collection Tube and centrifuge for 15 seconds.

7. Discard the flow-through and place the column back into the same Collection Tube.8. Add 200 μL of Endo-Wash Buffer to the column. Centrifuge for 30 seconds. It is not necessary to empty the collection tube.

9. Add 400 µL of Zyppy™ Wash Buffer to the column. Centrifuge for 1 minute.10. Transfer the column into a clean 1.5 mL microcentrifuge tube then add 30 µL of Zyppy™ Elution Buffer 2 directly to the column matrix and let stand for one minute at room temperature.

11. Centrifuge for 30 seconds to elute the plasmid DNA

Faster Better Biotech: Plasmid Yield

How do you check to see if you got plasmid? Old way…waste the small sample in restriction digest or UV spec.

Now…Use a G-Biosciences Nucleic dotMetric™ Assay kit (Geno Technology Inc) to quantify the amount of DNA in the plasmid sample. Take a 1 μL sample and blot it on the test strip.

Then using their DNA nucleic acid indicator and washes, stain the DNA sample proportional to the mass present.

G-Biosciences Nucleic dotMetric Assay 1 µl Assay For Estimation of DNA Concentration (< 1

µg/µL)

1. Dilute samples with Nucleic Acid Dilution Buffer (1:1 to 1:10 fold)

2. Apply 1-5 µl of each dilution onto the NUCLEIC Test Strip (remove the protective cover 1st)

3. Developed with about 500 µL of NUCLEIC Dye (cover all dots) about 1-2 minutes. Remove dye and wash/dunk in dH20

4. Compare the color density of the nucleic acid spots to the NUCLEIC standard strip.

No spectrophotometers or cuvettes required.

Faster Better Biotech: DNA Band Visualization

1. Run samples that contain µg amounts of (pAmy) DNA on agarose gels

2. Can run make TAE agarose gels (slow way) or use LB buffer to make and run gels….faster, better way.

Faster, Better Biotech:DNA (Agarose) Gels

Gels made with TAE Buffer

A tris acetate EDTA or tris borate EDTA gel tolerates only 110 volts, takes three times as long to run, and results in fuzzy DNA patterns.

Tris also produces loads of unwanted heat - heat that limits the voltage that can be used.

•Slow gel runs of 45-60 min•Steams up gel box

Gels made with Lithium Borate (LB) Bufferhttp://www.fasterbettermedia.com

Lithium borate conducts electricity better than TRIS so you can turn up the voltage and current on agarose gels :

•Fast gel run times (8-15 min)•Same protocol as TAE gels just substitute 1X LB buffer for 1X TAE•Purchase as 10X or 20X concentrate

pAmy VisualizationUse 1X LB buffer/0.8% agarose gel with 2-6 well combs.

Have only 1 cm of running buffer over gel.

1. Add 2 µL of EZ-Vision Loading Dye to 10 µL of sample.

2. Load all 12 µL of each sample to a well. Have at least one lane of DNA standard markers (Record what is loaded in each well.

3. Run gel for 10-15 min at 300 V (or as close as you can get)

4. Transfer gel to UV light box and visualize. Photograph.Uncut pAmylase plasmid is about 7,000 bp.

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