Functional Genomics Research Stream. Saccharomyces sensu stricto. Aseptic Technique III III

Transcription

1 Genomics Research Agenda IV Enzymatic Assays Functional Genomics Research Stream Research Meeting: February 1, 2011 Aseptic Technique, Solid & Liquid Media Cell Growth Laboratory Notebooks, Micropipettes Saccharomyces sensu stricto Saccharomyces paradoxus Saccharomyces cerevisiae Saccharomyces mikatae Saccharomyces bayanus III Molecular Preparations III Aseptic Cell Culture Safety and Basic Operations 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. II I

2 Saccharomyces Growth Solid Media (Plates) Liquid Media (Cultures) Both made from YPD Yeast Extract Peptone (animal protein) Dextrose (carbohydrate) Solid Media Growth Wikipedia Using Solid Media Streak One Other Representation Streak Two From Course Textbook (Wiley) Streak Three Streak Four

3 Yeast Growth Flow Liquid Media Growth colony dilute to ~0.2 Streaked Plate Overnight Culture overnight Large Culture during day Cell Culture Dilutions Name Definition Known? Cell Culture V1 Overnight Culture Volume Needed No? S1 Overnight Culture OD Yes OD 3.5 V1S1 = V2S2 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 diluted 1/5 to measure 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 ml of YPD to make 50 ml culture at OD 0.2.

4 Spectrophotometry Spectronic 20D+ Cell Culture Quantitation by Spectrophotometry Light Source λ I Measured Absorption λ Cell Culture (in cuvette) Transmission = 100%! I / Absorbance = -log (I / ) incident on sample I makes it through sample Good News: Space Age cuvettes,! 3 ml required Bad News:1960 s Space Age Doubling Times A Growth Curve Yeast: ~ 1.5 hours Let us say you start a culture OD When OD hours later? When OD hours later? When OD hours later? When OD hours later?

5 Growth Curves Examined OD600 ~ 0.80 Solid Media Growth Characterization OD600 ~ 0.2 Current Protocols in Essential Laboratory Skills Plating for Growth Defect (serial dilution left to right) Plating for Growth Defect (serial dilution left to right) 1/1 1/10 1/100 1/1000 Strain A Strain B Strain C Strain D Strain E Hu, Killion, Iyer Nature Genetics 2007

6 Plate Spotting Setup Grown at 30 C Grown at 37 C Serial Dilution 1 ml 0.9 ml 0.9 ml 0.9 ml S288C transfer 1 ml transfer 1 ml transfer 1 ml mix well mix well mix well OTHER 1/1 1/100 1/10 1/1000 same plating setup 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 Tradition, Practicality, Legality Properties: Hard-bound, Long Life Paper Laboratory Storage All Data Documented Chapter 4 of Online Course Notebook Numbering, Sequential, Chronological All Processes Documented (Repeat)

7 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. Lab Equipment 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? Micropipettes Volume Lock Ring Plunger Volume Readout Multi-Channel Disposable Tip 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

8 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 Stream Issues For Micropipette L 1000 Volume Range: 100 to 1000 µl Results Central

9 Academic Issues RPR I grades posted on Blackboard 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 I due The Semester spring break RPR I RPR II RPR III II&III due RPR IV spring break spring break through rest of semester... Laboratory Log Micropipette Technique