Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of...
-
Upload
ashley-long -
Category
Documents
-
view
215 -
download
0
Transcript of Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of...
![Page 1: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/1.jpg)
![Page 2: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/2.jpg)
Lab safety
• No food, backpacks in lab
• No open-toed shoes
• Goggles
• Make sure you know the location of safety equipment
• Buddy system
![Page 3: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/3.jpg)
Notebooks(p.4)
• No dictated format (bound?)
• Goals: – replicate the experiment
• Procedures• Real-time modifications of procedures
– understand outcomes• Observations• Raw data• Interpretation of data
![Page 4: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/4.jpg)
Notebooks
• Title
• Purpose
• Procedures– Calculations, etc.
• Observations– Raw data, processed data
• Conclusions
![Page 5: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/5.jpg)
Lab reports(see due date on p. 1)
• Two “short” reports– Experimental procedures & Results sections
• Several sets of DAQs
• One “formal” report– JBC-style (Abstract, intro, exp proc, etc.)– Includes two due dates: rough draft & final
• Experimental presentation
![Page 6: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/6.jpg)
Short reports(see p. 5)
• Title– Appropriate to the research being done
• Bad title: “Introduction to biochemical techniques”
![Page 7: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/7.jpg)
Short reports
• Materials & Methods/Experimental Procedures – Terse, to-the-point– Someone else should be able to replicate the
experiment– Concise vs. thorough– Passive tense
![Page 8: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/8.jpg)
We obtained Bradford reagent from the refrigerator at the northend of room 207. We added 2.0 mL of Bradford reagent to plastic cuvettes. Paul added the protein samples containingBovine serum albumin into the reagent. Dave inserted the cuvettes into the sample holder in the Cary UV/Vis spectrophotometer, and Mary pressed the button to read the absorbancy at 595 nm.
![Page 9: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/9.jpg)
Aliquots (50 L) of bovine serum albumin (BSA) standardsor the unknown sample were added to 2.0 mL Bradford reagent in plastic cuvettes. After a five minute incubationat room temperature, the absorbances at 595 nm were determined with a UV/Vis spectrophotometer (Varian, Inc.).
![Page 10: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/10.jpg)
Short reports
• Results– Narrative (still passive tense)– More description of motivation– Assume a relatively naïve reader
• You yesterday
![Page 11: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/11.jpg)
Short reports• Difficulties
– Determining what numbers go where• ie. Making a standard curve: do you need to say in the M&M
that you made standards of 0.05, 0.1, 0.2, 0.3, etc. g/mL BSA?
• Results? Figures?
– Concise vs. thorough– Organization
• Hint: use subtitles
– Scientific language• The final yield was pretty good.
– Hint #2: Look at JBC articles!
![Page 12: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/12.jpg)
Techniques
• Spectrophotometers• Micropipettors
![Page 13: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/13.jpg)
Pipettors
• Fragile/easy to break!
• Can be very accurate – can be very inaccurate
![Page 14: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/14.jpg)
Pipettors
• Basic features– Disposable tip
• Cross-contamination
– Dial• Set volume
– Plunger– Eject button (tip discard)
![Page 15: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/15.jpg)
Pipettors
• Appropriate volumes– 0.5 – 10l (P10)– 10-100 (P100)– 100-1000 (P1000) (1000 l = 1ml)– 1000-5000
• More accurate closer to upper limit– eg. use P100 for 100L volumes
![Page 16: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/16.jpg)
Pipettors
• How to choose volume
• How to seat tip
• Plunger ‘stops’– Top– 1st push– 2nd push (expel)
![Page 17: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/17.jpg)
Pipettors
• Watch tip– Air bubbles– Liquid on tip (drops carried over)– Liquid in tip (remaining after release)
• Dispensing– Into empty container: against the side– Into “full” container: immerse tip
![Page 18: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/18.jpg)
Pipettors
• Sources of error– Tip not fully seated– Air bubble (not enough volume)– Drop on outside (too much volume)– Liquid remaining (not enough volume)– Too slow for time sensitive experiment?
![Page 19: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/19.jpg)
Spectrophotometry
![Page 20: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/20.jpg)
Spectrophotometry
• Light absorbed by a sample– Depends on:
• Concentration of absorbing species (how much)• Path length (machine – constant)• ‘Molar absorptivity’ (identity of absorbing species)
A = εCL
Final result
Easily measurable (1 cm)Standardcurve
![Page 21: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/21.jpg)
Determining protein concentrations
• 0.5 mg/ml = 0.5 g/l
• 0.5 mg/ml = 500 g/ml
![Page 22: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/22.jpg)
Determining protein concentrations
• Direct: protein itself is the light-absorbing species– UV absorbance by peptide bond (amide)
• ~220 nm
– UV absorbance by aromatic functional groups• ~280 nm
– Non destructive– Lower sensitivity– Interference by other compounds– Protein content (A280)
• eg. protein with zero Y, W amino acids will absorb little or no light at A280
![Page 23: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/23.jpg)
Determining protein concentrations
• Indirect– Absorbance by
environmentally-sensitive dye
– Dye changes color when bound to protein
– Higher sensitivity, reproducibility (Vis)
– Cheap & easy– Destructive: sample no
longer useable
![Page 24: Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system.](https://reader030.fdocuments.net/reader030/viewer/2022032722/56649ce45503460f949b03e9/html5/thumbnails/24.jpg)
Determining protein concentrations
• Bradford dye (Coomassie Brilliant Blue)– Red, no protein– Blue, protein-bound
• Which wavelength to use?
• Binds to positive charges– How universal is a standard curve?