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Calculating Primer Quantity
Conversion to Absolute Quantity (in pmole)
| Primer in pmol = |
Weight in µg x 1,000,000
Length x 327 |
Example: 0.1 µg of 20-Oligomer:
0.1 x 1,000,000
20 x 327 |
= 15.3 pmol Primer |
Conversion to Weight (in µg)
| Weight in µg = |
pmol x Length x 327
1,000,000 |
Example: 10 pmol of 25-Oligomer:
10 x 25 x 327
1,000,000 |
= 0.081 µg Primer |
Calculation Molar Concentration of the Primer
Micromolar concentration of primer = pmoles/µl
Example 1: 20 pmoles of primer in 100 µl PCR mixture = 0.20 micromolar
(µM)
Example 2: Primer is 24 nucleotides in length and is dissolved in 0.1
ml of water
A 10 µl aliquot is diluted to 1.0 ml for A260
measurement: A260 = 0.76
The stock solution has an absorbance at 260 nm (A260)
of 76
The stock solution (0.1 ml) contains 2.6 A260
units
The base composition of the primer is: A = 6, C = 6, G = 6, T = 6
The Molar Extinction Coefficient at 260 nm for the primer = k (15,200)
+ l (12,010) + m (7,050) + n (8,400)
Where: k = number of A's; m = number of C's; l = number of G's; n =
number of T's
The Molar Extinction of the PCR Primer = 6 (15,200) + 6 (12,010) + 6
(7,050) + 6 (8,400) = 255,960
The Molar Concentration of the PCR primer stock solution is:
76
255,960 |
= 297 micromolar |
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