Functional Genomics Research Stream. Lecture: February 17, 2009 Masses, Volumes, Solutions & Dilutions

Transcription

1 Functional Genomics Research Stream Lecture: February 17, 2009 Masses, Volumes, Solutions & Dilutions

2 Agenda Lab Work: Last Week New Equipment Solution Preparation: Fundamentals Solution Preparation: How To Dilution Principles Lab Work: This Week

3 Lab Work: Last Week Solution 1 Solution 2 Solution 3 Not Water Water Water Which volume had the capacity to show the difference? 25 µl, 100 µl, 500 µl Take Home Lesson I: Stop & Think About It Take Home Lesson II: Conclusions = Lab Notebook

4 Equipment Update Lectures will progressively cover new equipment. Always updated: Appendix II - Laboratory Equipment Online Course Notebook Microsoft Excel Tutorial General lab principle: Read then ask.

5 Metric Prefixes Review

6 Pipettes & Bulbs

7 Beakers, Flasks, Media Bottles

8 Mixers & Stir Bars Mixing Apparatus Magnetic Sterile? Heat

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10 Vortexers & Water Baths Vortexers mixing reagents lysing cells Water Baths constant heat application

11 Laboratory Water Deionized Water di-h2o Distilled Water dd-h2o

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

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

14 SDS MSDS Continued Other Properties: Personal Protection 20 C + Temperature Helps

15 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.

16 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 (not this week). 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.

17 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.

18 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 (taught next week). 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, 2/17/2009.

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20 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)

21 Expressing Concentration 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)

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

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

24 Weight / weight (w/w) % Ratio of weight of solute to total weight of the solution (not solvent) multiplied x 100. If 150 g aqueous NaCl contains 25 g solid NaCl reagent: 25/150 * 100 = 16.7 (w/w) %

25 Weight / volume (w/v) % Ratio of weight of a solute to the total volume of solution (not solvent) multiplied by 100. If 150 ml aqueous NaCl contains 25 g solid NaCl reagent: 25/150 * 100 = 16.7 (w/v) % Note: units of numerator and denominator differ - still very convenient.

26 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) %

27 Molarity (M) Very widely used unit of concentration. Number of moles per 1 L solution. NaOH Example: molecular weight = 40 g/mol 1L of 1M NaOH contains 40 g NaOH (dry)

28 Basic Dilution Principles Common to Make Stocks: High Concentrations Reduced Variability of Experiments Clean Dilution Needed for Experimentation

29 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

30 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 150mM dilution.

31 Serial Dilutions Goal = Series of Dilutions 1/1 1/10 1/100 1/1000 etc...

32 Serial Dilution 10 ml 9 ml 9 ml 9 ml transfer 1 ml transfer 1 ml transfer 1 ml mix well mix well mix well reagent water water water

33 Serial Dilution: Result 9 ml 9 ml 9 ml 10 ml 1/1 1/10 1/100 1/1000 reagent reagent reagent reagent

34 Assignment Five Purpose: Single Component Solution Preparation Multi Component Solution Preparation Dilution Series Laboratory Notebooks (always) Due: Tuesday, February 24

35 Laboratory Hours Lab hours will be logged this week 6 hours per week, cumulative Appendix I Laboratory Hours sign in, sign out full explanation Lab schedule on course calendar

36 Questions?