Preparing Solutions

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

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

Getting started  Beaker larger than final volume  Add 2/3 final volume of gdH 2 O

Add a magnetic spin bar

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

Issues  Heating  pH  Solvents  Noxious and hazardous compounds

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

“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)

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

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

Issues – “Begin with the end in mind”  Autoclaving  Filtering  Light  Heat  Containers

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

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

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

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

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

Additions from concentrated stock solutions  C 1 is the concentration of your stock solution  C 2 is the concentration you want in the end  V 2 is your final volume (q.s.)  V 1 is how much to add! C 1 V 1 = C 2 V 2

Additions from concentrated stock solutions  Make sure concentration units are the same (M, mM, %, etc.)  Make sure volumes are in the same units (l, ml, etc.)  Solve for V 1  C 2 cannot be bigger than C 1  V 1 cannot be bigger than V 2 C 1 V 1 = C 2 V 2

X solutions  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 C 1 V 1 = C 2 V 2 to determine amount needed (V 1 ) of concentrated stock (C 2 )

A Note on Buffers  Buffer refers to a weak acid or base and their salt 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

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

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 gdH 2 O  Add 6 M HCl dropwise to adjust pH to 7.9  Bring to 500 ml in graduated cylinder