Preparing Solutions

1. Preparing Solutions

2. Short Form • Obtain the required amount of ingredients • Dissolve them • Bring to volume (q.s.) • Store

3. Short Form • Obtain the required amount of ingredients • Dissolve them • Bring to volume (q.s.) • Store

4. Getting started • Beaker larger than final volume • Add 2/3 final volume of gdH2O

5. Add a magnetic spin bar

6. Begin adding reagents • Use a clean spatula and weigh dish for each ingredient • Never return excess material to its container

7. Issues • Heating • pH • Solvents • Noxious and hazardous compounds

8. Short Form • Obtain the required amount of ingredients • Dissolve them • Bring to volume (q.s.) • Store

9. “Finishing” a solution • Everything should be fully dissolved* • Temperature must be cool enough to handle. • pH must be set • Transfer to graduated cylinder and bring to final volume • Final volume = q.s. (quantum satis)

10. Late Edition • (Should be late addition) • Filter sterilized amendments • Heat sensitive, reactive (e.g. ampicillin) • 1000X – Volume insignificant

11. Short Form • Obtain the required amount of ingredients • Dissolve them • Bring to volume (q.s.) • Store

12. Issues – “Begin with the end in mind” • Autoclaving • Filtering • Light • Heat • Containers

13. Labels • Composition - 20X SSC (better exact composition) • (special) Storage conditions • Date Made (include the year!) • Your name • Autoclave tape

14. Short Form • Obtain the required amount of ingredients • Dissolve them • Bring to volume (q.s.) • Store

15. Powders – Molarity; Three numbers (mol l-1)(g mol-1)(l) = grams required • (g mol-1) MW, FW (hydrates), % purity, free acid vs. salt, etc.

16. Powders – % (w/v) 1% = g 100 ml-1 • Grams of powder added per 100 ml final volume (q.s. rules)

17. Liquids – Molarity; Density (mol l-1)(g mol-1)(l) = grams required • Divide g required by density of liquid (g ml-1) • Pipette this amount of liquid • Remember to account for purity.

18. Additions from concentrated stock solutions C1V1 = C2V2 • C1 is the concentration of your stock solution • C2 is the concentration you want in the end • V2 is your final volume (q.s.) • V1 is how much to add!

19. Additions from concentrated stock solutions C1V1 = C2V2 • Make sure concentration units are the same (M, mM, %, etc.) • Make sure volumes are in the same units (l, ml, etc.) • Solve for V1 • C2 cannot be bigger than C1 • V1 cannot be bigger than V2

20. 20X SSC Buffer 5X Wash Solution 10X RE Buffer 20X TAE Buffer 100X Vitamins 1000x Ampicillin X refers to relative concentration of some complex solution 1X is the normal working concentration Use C1V1 = C2V2 to determine amount needed (V1) of concentrated stock (C1) X solutions

21. A Note on Buffers • Buffer refers to the salt of a weak acid or base that helps to keep a constant pH. • Buffer is used to describe solutions containing pH buffers. • E.g. 10X PCR buffer contains 10 times of everything required for the PCR reaction including Tris, a pH buffer

22. Adding the Buffer • Tris, Acetate, Phosphate • Add buffer to give desired concentration • Add acid (or base) to adjust pH to desired value • Bring to volume

23. 0.5 l of 50 mM Tris-HCl, pH 7.9 • (0.05 mol l-1)(121.1 g mol-1)(0.5 l) = 3.03g • Add 3.03 g Tris (base) to about 400 ml of gdH2O • Add 6 M HCl dropwise to adjust pH to 7.9 • Bring to 500 ml in graduated cylinder

24. Multi Component Solutions...20% (wt/vol) sucrose-0.3 M Tris-HCl (pH 8)-1mM EDTA.... • Start with ca. 600 ml water • Add _____g sucrose, spin to completely dissolve • Add _____g Tris base, spin to completely dissolve • Add _____ml of 250 mM Stock EDTA pH 8 solution • Adjust pH to 8.0 with _____. • Bring to 1000 ml total volume.