Concentration
Take a little time to re-visit your chemistry and understand the conversions.
Another way of expressing concentration is called molarity. Molarity is the number of moles of solute dissolved in one liter of solution. The units, therefore are moles per liter, specifically it's moles of solute per liter of solution.
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molarity = |
moles of solute liter of solution |
Rather than writing out moles per liter each time, molarity is abbreviated as M or M. "Moles" is a measure of the amount or quantity of material. The number of molecules in one mole is equal to 6.0221367 x 1023. In contrast, "Molarity" measures the concentration of mole units in solution. Reagents in the lab give a molecular weight (abbreviated as M.W.), which is the amount of grams/mole (g/mol) for that product.
The most useful formula you will be using is:
M1 V1 = M2 V2,
Starting molarity (M1) multiplied by starting volume (V1) equals the desired molarity (M2) multiplied by the desired volume (V2).
Primer Resuspension
Primer information sent from a company will list the molecular weight of your primer (g/mol) and the amount of nmoles that they sent. If you have 29.4 nmol of primer and you want to make a 100 μM stock solution in TE – simply multiply the amount of nmoles by a factor of ten and add that many microliters. In this example add 294 μL.
Calculations
100 μM = 100 μmol/L = 0.1 nmol/μL
29.4 nmol X 1 μL/0.1 nmol = 294.0 μL
Our general practice is to make a 100 μM stock solution with TE and then dilute to a 10-20 μM working solution using Tris pH 8.3 or filtered ddH2O.
Confirm the concentration using the nanodrop, because the concentrations provided by the company are often wrong. To nanodrop a primer you must change the setting parameters to measure a single stranded template. You will also see DNA-33, which refers to the wavelength-dependent extinction coefficient in ng-cm/microliter:
• Double-stranded DNA: 50
• Single-stranded DNA: 33
• RNA: 40
The nanodrop gives readings in ng/µl, but you often need to convert this value into pmol/µl. If your primer has a molecular weight of 6000 g/mol, this is the same as saying 6000 ng/nmol, and the nanodrop reading is 9.6 ng/ul, here is how to convert the value topmol/µl:
9.6 ng/µl ÷ 6000 ng/nmol = 0.0016 nmol/µl
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Nucleotide concentrations
Nucleotides for PCR reactions are abbreviated as dNTPs, where the N refers to the four nuleotides (ATCG). They are usually shipped with each dNTP concentrated at 100 mM. They are suspended and diluted in filtered ddH2O. A standard PCR reaction uses 2 mM of each dNTP.
Calculations
To make working stocks of 100 μL:
M1V1 = M2V2
100 mM X V1 = 2 mM X 100 μL
V1 = 2 μL
Therefore, add 2 μL of each dNTP (2 μL X 4 dNTPs = 8 μL) into 92 μL of filtered ddH2O.