preparing solutions. short form obtain the required amount of ingredients dissolve them bring to...
Post on 19-Dec-2015
223 views
TRANSCRIPT
Begin adding reagents
Use a clean spatula and weigh dish for each ingredient
Never return excess material to its container
“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
Labels
Composition - 20X SSC (better exact composition)
(special) Storage conditions Date Made (include the year!) Your name Autoclave tape
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
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
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!
C1V1 = C2V2
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 V1
C2 cannot be bigger than C1
V1 cannot be bigger than V2
C1V1 = C2V2
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 C1V1 = C2V2 to determine amount needed (V1)
of concentrated stock (C1)
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
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 gdH2O Add 6 M HCl dropwise to adjust pH
to 7.9 Bring to 500 ml in graduated
cylinder
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.