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Quantitative Analysis of the PCR Reaction
PCR Conditions for Lambda Control:M
- 200 µM (each) dNTPs; total [dNTPs] = 0.8 mM
- Total [MgCl2] = 1.5 mM
- Free [MgCl2] = 0.7 mM
- 2.5 Units AmpliTaq® DNA Polymerase per 100 µL
- 1 micromolar (each) primers [PCR01, PCR02]
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- Bacteriophage Lambda DNA template
(Template ds DNA = 48,500 bp)
- Target = 500 bp
- Number of cycles = 25
- Buffer: 10 mM Tris-HCl, pH 8.3 (at 25 °C); 50 mM KCl
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| Before PCR |
After PCR |
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Weight |
Moles |
Molarity |
Molecules |
Weight |
Moles |
Molarity |
Molecules |
| Template (48,500 bp) |
1 ng |
3.10x10-17 |
3.10x10-13 |
1.86x107 |
1 ng |
3.00x10-17 |
3.00x10-13 |
1.81x107 |
Target
(500 bp) |
10 pg |
3.00x10-17 |
3.00x10-13 |
1.81x107 |
1 µg |
3.00x10-12 |
3.00x10-8 |
1.81x1012 |
| Primers (25-mers) |
1623 ng |
2.00x10-10 |
2.00x10-6 |
1.20x1014 |
1574 ng |
1.94x10-10 |
1.94x10-6 |
1.17x1014 |
| dNTPs |
39 µg |
8.00x10-8 |
8.00x10-4 |
4.82x1016 |
37 µg |
7.70x10-8 |
7.70x10-4 |
4.64x1016 |
| Magnesium Ion |
3.6 µg |
1.50x10-7 |
1.50x10-3 |
9.03x1016 |
3.6 µg |
1.50x10-7 |
1.50x10-3 |
9.03x1016 |
| AmpliTaq® DNA Polymerase |
12.5 ng |
1.33x10-13 |
1.33x10-9 |
8.01x1010 |
12.5 ng |
1.33x10-13 |
1.33x10-9 |
8.01x1010 |
Assumptions and Data:
- AmpliTaq® DNA Polymerase specific activity
= 250,000 Units/mg
- Average MW of a dNTP is 487 Daltons
- Average MW of a dNMP is 325 Daltons
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- Achieve at least 105-fold amplification
- AmpliTaq® DNA Polymerase half-life is not considered
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Spectrophotometric Conversions:
Double-stranded DNA (ds DNA):
A260 = OD260 = 1 for a 50 µg/mL solution
Single-stranded DNA (ss DNA):
A260 = OD260 = 1 for a 33 µg/mL solution
RNA: A260 = OD260 = 1 for a 40 µg/mL solution
Reference: Freifelder, D., Physical Biochemistry: Applications to
Biochemistry & Molecular Biology, W.H. Freeman and Company, CA,
1982, p. 494-536.
Useful Equations and Nucleic Acid Molecular Weight Data:
Absorbance =
Molar Extinction Coefficient x Concentration x Pathlength
500 bp of double-stranded DNA = 325,000 Daltons
500 nt* of single-stranded DNA = 162,500 Daltons
Average MW of dNMP is 325 Daltons
(*nt = nucleotide)
Oligomer Quantitation:
For a 20-mer, a stock solution with A260 = 1 contains 5 nmol
5 nmol = 33 µg/(20 x 325)
For a 40-mer, a stock solution with A260 = 1 contains 2.5
nmol
2.5 nmol = 33 µg/(40 x 325)
Conversion of pmoles of primer to µg of primer:
Multiply pmoles by (length x 325)/1,000,000
Example: 10 pmoles of a 25-mer
(10 x 25 x 325)/1,000,000 = 0.081 µg primer
Conversion of µg of primer to pmoles of primer:
Multiply by 1,000,000/(length x 325)
Example:
0.1 µg of a 20-mer
(0.1 x 1,000,000)/(20 x 325) = 15.4 pmoles primer
Calculating Primer Concentrations for PCR Amplification:
Micromolar concentration of primer = pmoles/µL
Example 1:
20 pmoles of primer in 100 µL PCR mixture =
0.20 micromolar
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 = OD260 = 0.76
The stock solution has an absorbance at 260 nm (A260) of 76
The stock solution (0.1 mL) contains 7.6 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 = a(16,000)
+ b(12,000) + c(7,000) + d(9,600)
where: a is the number of A's, b is the number of G's, c is the number
of C's, d is the number of T's
The Molar Extinction Coefficient of the PCR primer is:
6(16,000) + 6(12,000) + 6(7,000) + 6(9,600) = 267,600
The Molar Concentration of the PCR primer stock solution is:
76/267,600 = 284 micromolar
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