Functional Genomics Research Stream. Research Meeting: February 7, 2012 Reagent Production, Buffers & ph

Transcription

1 Functional Genomics Research Stream Research Meeting: February 7, 2012 Reagent Production, Buffers & ph

2 Section VII Yeast Growth Flow Wednesday Thurs. morning S288C S288C colony dilute to ~0.2 S288C other Streaked Plate other Overnight Culture overnight other Large Culture during day

3 Plate Spotting Setup Thurs. morning S288C Grown at 30 C Grown at 37 C OTHER OD = 1.0 1/1 1/100 1/10 1/1000 same plating setup

4 Section VII Yeast Growth Flow Wednesday Thurs. morning colony Streaked Plate Overnight Culture overnight

5 Serial Dilution OD = 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

6 Serial Dilution: Result OD = ml 0.9 ml 0.9 ml 1.0 ml 1/1 1/10 1/100 1/1000 culture culture culture culture

7 High Purity Reagents Dry Form Chemicals Highly Pure ACS Grade Solutions & Reagents Made by Mixing Avoid Contamination

8 MSDS Information Material Safety Data Sheets Product & Laboratory Safety Search Many Online Sources SDS Example: Molecular Weight Chemical Formula - CH3(CH2)11OSO3Na

9 Expressing Concentration Weight per Unit Volume Percent Composition Weight / weight (w/w) Weight / volume (w/v) Volume / volume (v/v) Normality (N) Molarity (M) Molality (m) Mole Fraction (X)

10 Weight per Unit Volume Mass per Volume Mass divided by Volume g / L mg / ml µg / µl

11 Percent Composition Weight / weight (w/w) % Weight / volume (w/v) % Volume / volume (v/v) %

12 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.

13 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

14 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

15 Basic Dilution Principles Common to Make Stocks Higher concentrations... Reduced variability of experiments... Clean... Efficient... Dilution Needed for Experimentation

16 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

17 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 V = 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.

18 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.

19 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.

20 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.

21 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). many reagents will not go into solution w/o ph 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, 2/8/2011.

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23 Synthesis in Conical Tubes

24 Conical Tubes: Example 1. Making sodium acetate, 1M. Acquire sodium acetate. 2. Experiment calls for 1M sodium acetate. 3. Experiment needs 1 ml; we ll make 10 ml for repeats. 4. Mass Decision: Molecular Weight = g/mol; need 0.82 g for 10 ml. 5. Volume Decision: Will store in 15 ml conical tube (15 ml capacity). 6. Weight out 0.82 g of sodium acetate.

25 7. Acquire 15 ml conical tube. 8. Add 8.5 ml di-h2o to the conical tube. 9. Add the 0.82 g of sodium acetate. 10. Vortex, vortex, vortex. 11. Allow to mix completely. 12. ph adjustment performed (if needed). many reagents will not go into solution w/o ph 13. Transfer to volumetric measuring cylinder. 14. Complete the solution by bringing volume up to 100% of calculated volume (~10 ml). 15. Transfer to storage container (media bottle). 16. Tape label completely: 1M sodium acetate, 2/8/2011.

26 Single Component from Stock Stock: 100 mm Tris Need: 1mL of 10 mm Tris Process: V1S1 = V2S2 Tris V1S1 = V2S2 V1(100) = 1(10) V1 = 0.10 ml Thus, add 0.10 ml of 100 mm Tris to 0.90 ml sterile water = 1 ml 10 mm Tris

27 Multi Component from Stocks Stock: 100 mm Tris, 100 mm NaCl Need: 1mL of (10 mm Tris, 10 mm NaCl) Tris V1S1 = V2S2 V1(100) = 1(10) V1 = 0.10 ml NaCl V1S1 = V2S2 V1(100) = 1(10) V1 = 0.10 ml Thus, add 0.10 ml of 100 mm Tris to 0.10 ml of 100 mm NaCl and then add 0.80 ml sterile water = 1 ml (10 mm Tris, 10 mm NaCl)

28 Hydrogen and Hydroxide Ions Pure water dissociates (very weakly) as follows: H2O H + + OH - [H + ] = 10-7 M [OH - ] = 10-7 M If I were to add 1 ml of HCl to 1 L of H2O HCl H + + Cl - H2O H + + OH - [H+ ] = 10-3 M [OH - ] = M

29 ph Pure water dissociates (very weakly) as follows: H2O H + + OH - [H + ] = 10-7 M [OH - ] = 10-7 M If I were to add 1 ml of HCl to 1 L of H2O HCl H + + Cl - ph = 3 H2O H + + OH - ph = -log [H + ] ph = 7 [H+ ] = 10-3 M [OH - ] = M

30 Buffer Solutions Can be made of a weak acid and its conjugate base: HA H + + A - Dissociation constant: Ka= [H+ ][A - ] [HA] If I were to add HCl to HA buffer solution HCl H + + Cl - HCl + HA H + + A - + Cl -

31 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

32 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

33 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

34 Laboratory Issues

35 Laboratory Notebook Evaluation Points Possible Points Earned Comments CONTENT 50 Could I recreate your experiments - EXACTLY - as you did them by simply following along in your notebook? Can I tell what your are doing and why you are doing it? TIMES & DATES 25 Can I tell when you started work and the hour-to-hour, day-to-day nature by which it flowed? Are there huge gaps that make no sense? Are experiments out of order? LABELS 25 Are all figures, gels and tables clearly and completely labeled? Would I know how to interpret any figure without asking you? Late or Missing Notebook Penalty: - Final Points: 100