Functional Genomics Research Stream. Yeast Growth Flow. High Purity Reagents III III

Transcription

1 Genomics Research Agenda IV Enzymatic Assays Functional Genomics Research Stream Molecular Preparations Aseptic Cell Culture Research Meeting: February 8, 2011 Safety and Basic Operations Reagent Production, Buffers & ph Yeast Growth Flow colony Streaked Plate overnight High Purity Reagents Dry Form Chemicals Highly Pure ACS Grade Solutions & Reagents dilute to ~0.2 Overnight Culture III Made by Mixing Large Culture during day Avoid Contamination III II I

2 MSDS Information Material Safety Data Sheets Product & Laboratory Safety Search Many Online Sources SDS Example: Molecular Weight Chemical Formula - CH3(CH2)11OSO3Na 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) Weight per Unit Volume Percent Composition Mass per Volume Mass divided by Volume g / L mg / ml µg / µl Weight / weight (w/w) % Weight / volume (w/v) % Volume / volume (v/v) %

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

4 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. 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.

5 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). 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. Synthesis in Conical Tubes

6 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. 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. 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 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 0.90 ml sterile water = 1 ml 10 mm Tris 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)

7 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 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 Laboratory Issues 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

8 Announcement: New RPR III! Media Bottles Haiku life so wonky dark in the drawer, leaky and gross shelf life is for me 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 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 Are all figures, gels and tables clearly and completely labeled? Would I know how to interpret any figure without asking you? Plate Contaminations & Issues Late or Missing Notebook Penalty: - Final Points: 100

9 30C Saccharomyces cerevisiae Saccharomyces paradoxus 37C

10 Lab Progress Continue Research Progress Report II & III Consider Timing, Constraints - Prepare Do Not Procrastinate Do Not Abuse Freedom & Responsibility Lab Open & Mentors Present 10am to 8pm (M, W, Th, F) No Tuesdays For Now... Research Resource Calendars... 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