Functional Genomics Research Stream Research Meeting: January 31, 2012 Aseptic Technique, Solid & Liquid Media Cell Growth Laboratory Notebooks, Micropipettes
Aseptic Technique Prevents media, cultures, stock reagents from becoming contaminated. Also called sterile technique. Patterns of habit, movement, and prevention. Very difficult to know if you have good aseptic technique; confounding variables.
Saccharomyces Growth Solid Media (Plates) Liquid Media (Cultures) Both made from YPD Yeast Extract Peptone (animal protein) Dextrose (carbohydrate) Wikipedia
Solid Media Growth
Using Solid Media Streak One Streak Two Streak Three Streak Four http://www.madsci.org/~lynn/micro/techniques/streaking/
Other Representation From Course Textbook (Wiley)
Liquid Media Growth
Yeast Growth Flow colony dilute to ~0.2 Streaked Plate Overnight Culture overnight Large Culture during day
Cell Culture Dilutions V1S1 = V2S2 Name Definition Known? Cell Culture V1 Overnight Culture Volume Needed No? S1 Overnight Culture OD Yes OD 3.5 diluted 1/5 to measure V2 Dilution Volume Yes 50 ml S2 Dilution Concentration Yes OD 0.2
Name Definition Known? Cell Culture V1 Overnight Culture Volume Needed No? S1 Overnight Culture OD Yes OD 3.5 V2 Dilution Volume Yes 50 ml S2 Dilution Concentration Yes OD 0.2 V1 S1 = V2 S2 V1 3.5 = 50 0.2 V1 = 2.85 ml Conclusion: We add 2.85 ml of overnight OD 3.5 culture to 47.15 ml of YPD to make 50 ml culture at OD 0.2.
Spectrophotometry Light Source λ Io Io Io Io Io I Measured Absorption λ Cell Culture (in cuvette) Transmission = 100% I / Io Absorbance = -log (I / Io) Io incident on sample I makes it through sample
Spectronic 20D+ Cell Culture Quantitation by Spectrophotometry cuvettes, 3 ml required Good News: Space Age Bad News:1960 s Space Age
Doubling Times Yeast: ~ 1.5 hours Let us say you start a culture OD600 0.10. When OD600 0.20 hours later? When OD600 0.40 hours later? When OD600 0.80 hours later? When OD600 1.60 hours later?
A Growth Curve
Growth Curves Examined OD600 ~ 0.80 OD600 ~ 0.2 Current Protocols in Essential Laboratory Skills
Solid Media Growth Characterization
Plating for Growth Defect (serial dilution left to right) Strain A Strain B Strain C Strain D Strain E 1/1 1/10 1/100 1/1000
Plating for Growth Defect (serial dilution left to right) Hu, Killion, Iyer Nature Genetics 2007
Plate Spotting Setup Grown at 30 C Grown at 37 C S288C OTHER 1/1 1/100 1/10 1/1000 same plating setup
Serial Dilution 1 ml 0.9 ml 0.9 ml 0.9 ml transfer 1 ml transfer 1 ml transfer 1 ml mix well mix well mix well culture media media media
Serial Dilution: Result 0.9 ml 0.9 ml 0.9 ml 1.0 ml 1/1 1/10 1/100 1/1000 culture culture culture culture
Laboratory Notebooks Section A2 of Course Textbook Chapter 4 of Online Course Notebook Tradition, Practicality, Legality Properties: Hard-bound, Long Life Paper Numbering, Sequential, Chronological Laboratory Storage All Data Documented All Processes Documented (Repeat)
1. Title & Date Title: One-line summation of work Date: Time and date of when procedure performed. 2. Rationale & Purpose A few sentences describing why you are doing the experiment. Details are important here. Which strains are you using? Is there an expected result? Is this a re-attempt from a previous experimental failure? Are any special experimental parameters being used for the first time or tests? 3. Expected Data Tables If you are collecting data during the procedure it is very good to include a table predicting how the data will be collected. Often this is not full size - it s just a small estimate of what the actual data tables will look like. 4. Actual Data Tables You have two choices when it comes to data tables. You can either populate the ones you created in Expected Data Tables or create new ones. 5. Procedure(s) A complete, step by step, every detail included account of what you did and how you did it for the experimental procedure. It is acceptable to include Xerox copies of standard protocols. It is also acceptable to denote or reference standard protocols such as Total RNA Preparation from FG website. You should not waste a lot of time writing the procedure in your notebook step-by-step. Remember - the goal is to reproduce your work. Denote information needed. 6. Results & Conclusions The results of the process. How did things work? Was the overall procedure successful? Will you need to do it again? Where does this procedure lead next?
Lab Equipment
Micropipettes Volume Lock Ring Plunger Volume Readout Disposable Tip Multi-Channel
Micropipette L 2 Volume Range: 0.2 to 2 µl Lowest Setting: 020 Highest Setting: 200 Readout: 023 would mean 0.23 µl Readout: 123 would mean 1.23 µl Micropipette L 20 Volume Range: 2 to 20 µl Lowest Setting: 020 Highest Setting: 200 Readout: 023 would mean 2.3 µl Readout: 123 would mean 12.3 µl Micropipette L 200 Volume Range: 20 to 200 µl Lowest Setting: 020 Highest Setting: 200 Readout: 023 would mean 23 µl Readout: 123 would mean 123 µl Micropipette L 1000 Volume Range: 100 to 1000 µl Lowest Setting: 020 Highest Setting: 100 Readout: 023 would mean 230 µl Readout: 099 would mean 990 µl Readout Example 0 2 2 3 4
For Micropipette L 2 Volume Range: 0.5 to 2 µl For Micropipette L 20 Volume Range: 2 to 20 µl For Micropipette L 200 Volume Range: 20 to 200 µl For Micropipette L 1000 Volume Range: 100 to 1000 µl
Stream Issues Caps on culture tubes - the Goldilocks zone How do we dispose of plates? liquid media? Saturday lab?
Academic Issues Continue Research Progress Report II Consider timing & constraints... Do not procrastinate... Do Not Abuse Freedom & Responsibility Lab open & mentors present 10am to 8pm (M, W, Th, F), calendar scheduling Tuesdays for plating and overnight cultures... Please see me about any concerns... lab unfamiliarity, new to research, traditional labs
The Semester I due RPR I RPR II RPR III RPR IV II&III due spring break spring break spring break through rest of semester...
Laboratory Log
Micropipette Technique