Laboratory Issues. Functional Genomics Research Stream. Research Progress Report II Issues. Media Bottles Haiku

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1 Functional Genomics Research Stream Laboratory Issues Research Meeting: February 9, 2010 Reagent Production, ph & Research Report III Concepts Media Bottles Haiku life so wonky dark in the drawer, leaky and gross shelf life is for me Research Progress Report II Issues

2 Yeast Growth Flow Lessons? colony dilute to ~0.2 Streaked Plate Overnight overnight Large during day Yeast Growth Flow Lessons? Yeast Growth Flow Lessons? colony dilute to ~0.2 colony dilute to ~0.2 Streaked Plate Overnight overnight Large Streaked Plate Overnight overnight Large during day during day

3 Growth Curves Examined OD600 ~ 0.80 Continued Laboratory Principles OD600 ~ 0.2 Current Protocols in Essential Laboratory Skills Laboratory Water Deionized Water di-h2o Distilled Water dd-h2o High Purity Reagents Dry Form Chemicals Highly Pure ACS Grade Solutions & Reagents Made by Mixing Avoid Contamination

4 MSDS Information Material Safety Data Sheets Product & Laboratory Safety Search Many Online Sources SDS Example: Molecular Weight Chemical Formula - CH3(CH2)11OSO3Na Accuracy in Measurement Volume Micropipette ( µl) Pipette (1 ml - 50 ml) Mass Analytical Balance (0.1 mg - 60 g) Top Loading Balance (0.01 g g) Expressing Concentration Weight per Unit Volume Weight per Unit Volume Percent Composition Weight / weight (w/w) Weight / volume (w/v) Volume / volume (v/v) Parts Per Million (ppm) Normality (N) Molarity (M) Molality (m) Mole Fraction (X) Mass per Volume Mass divided by Volume g / L mg / ml µg / µl

5 Percent Composition Weight / weight (w/w) % Weight / volume (w/v) % Volume / volume (v/v) % Weight / volume (w/v) % Ratio of weight of a solute to the total volume of solution (not solvent) multiplied by 100. If 100 ml aqueous SDS contains 20 g solid SDS reagent: 20/100 * 100 = 20 (w/v) % SDS Note: units of numerator and denominator differ - still very convenient. Volume / volume (v/v) % Ratio of the volume of liquid solute to the total volume of the solution (not solvent) multiplied by 100. When 70 ml of EtOH (ethanol) is diluted to a total volume of 100 ml: 70/100 * 100 = 70 (v/v) % EtOH Molarity (M) Very widely used unit of concentration. Number of moles per 1 L solution. NaOH Example: molecular weight = 40 g/mol (look up) 1L of 1M NaOH contains 40 g NaOH 1L of 100 mm NaOH contains 4 g NaOH

6 Basic Dilution Principles Common to Make Stocks Higher concentrations... Reduced variability of experiments... Clean... Efficient... Dilution Needed for Experimentation Basic Dilutions V1S1 = V2S2 Name Definition Known? NaCl V1 Stock Volume Needed No? S1 Stock Concentration Yes 1M V2 Dilution Volume Yes 100 ml S2 Dilution Concentration Yes 150 mm Name Definition Known? NaCl V1 Stock Volume Needed No? S1 Stock Concentration Yes 1M V2 Dilution Volume Yes 100 ml S2 Dilution Concentration Yes 150 mm V1! S1 = V2! S2 V1! 1000 = 100! 150 V1 = 15 ml Conclusion: We add 15 ml of 1M stock to 85 ml of di-h2o to make 100 ml of 150 mm dilution. Solution Making: Process 1. Acquire reagents. 2. Determine concentration(s) needed. 3. Determine appropriate volume(s) needed. 4. Mass Decision: Calculate reagent need(s) per concentration and volume decisions. 5. Volume Decision: Determine storage requirements (tube, flask, bottle). 6. Use appropriate balance to measure out needed reagent mass on weight boat.

7 7. Place chosen beaker on mixer. 8. Place clean mixer bar in beaker. 9. Add ~85% volume of water needed. 10. Start the mixer at a slow but functional speed. 11. Add the measured reagent to the mixing solution. 12. Allow to mix completely; apply heat if needed, safe. 13. Adjust the ph of solution. 14. Transfer to volumetric measuring cylinder. 15. Complete the solution by bringing volume up to 100% of calculated volume. 16. Transfer to storage container. 17. Label completely: solution name, concentration, date. Solution Making: Example 1. Making sodium acetate, 1M. Acquire sodium acetate. 2. Experiment calls for 1M sodium acetate. 3. Experiment needs 1 ml; we ll make 100 ml for repeats. 4. Mass Decision: Molecular Weight = g/mol; need 8.2 g for 100mL. 5. Volume Decision: Will store in glass media bottle (125 ml capacity). 6. Weight out 8.2 g of sodium acetate. 7. Will use small beaker capable of ~250 ml. 8. Place clean mixer bar in beaker. Place on mixer. 9. Add 85 ml di-h2o to the beaker. 10. Start the mixer at a slow but functional speed. 11. Add the 8.2 g of sodium acetate. 12. Allow to mix completely; apply heat if needed, safe. 13. ph adjustment performed (if needed). 14. Transfer to volumetric measuring cylinder. 15. Complete the solution by bringing volume up to 100% of calculated volume (100 ml). 16. Transfer to storage container (media bottle). 17. Tape label completely: 1M sodium acetate, 2/9/2010.

8 Synthesis in Conical Tubes ph negative log of dissolved hydrogen ions (H + ) ph adjustment of reagents ph in buffer systems Biological Buffers: Purpose Biological Example: Blood Biological systems tend to dislike significant shifts in ph. Our environment is full of weak acids and bases that can easily shift biological systems from a ph. Buffer systems prevent these shifts. Ideal blood ph ~ 7.4 As low as 6.8, death. As high as 7.8, death. CO2 and H + produced during processing of glucose.

9 Example: H2CO3 When blood ph rises, carbonic acid dissociates to form bicarbonate and H +. H2C03 HC H + When blood ph drops, bicarbonate binds H + to form carbonic acid. HC H + H2C03 Similar Laboratory Concerns (not so much death, rather ph shifts during experiments) Buffer Solutions Solutions used in a laboratory setting to control the ph of experimental settings. Goal = ph is held nearly constant. The ph is easily shifted by addition of: other reagents temperature evaporation Buffer Solutions: Examples Common Name pka at 25 C Buffer Range Mol. Weight Full Compound Name TAPS {[tris(hydroxymethyl)methyl]amino}propanesulfonic acid Bicine N,N-bis(2-hydroxyethyl)glycine Tris tris(hydroxymethyl)methylamine Tricine N-tris(hydroxymethyl)methylglycine HEPES hydroxyethyl-1-piperazineethanesulfonic acid TES {[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid MOPS (N-morpholino)propanesulfonic acid PIPES piperazine-n,n -bis(2-ethanesulfonic acid) Cacodylate dimethylarsinic acid MES (N-morpholino)ethanesulfonic acid Wikipedia, Buffer Solutions

10 Research Progress Report III