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PCR Tips, tricks, techniques and calculations
PCR Tips, tricks, techniques and calculations ~ overview
Information on the tabs is arranged thus:
- No. of cycles, Denaturation step, Annealing step and Elongation step
- an overview of the common rules and concentrations which should be observed when designing new experimental setups.
- Electrophoretic Resolution, Dye Migration,Physical Properties of Nucleotides, Biophysical Data for Deoxynucleoside Triphosphates and Conversion to Weight (Âµg/ml)
- Calculating primer quantity
- Genetic code, properties and molecular structure of amino acids
PCR Tips, tricks, techniques and calculations ~ Basic Rules
Step 1: No. of cycles
No. of source molecules
No. of cycles
50 and less
20-30, followed by a second PCR with "nestedâ€ť primers, i. e. with a primer pair which binds between the first two primers of the target sequence
Do not carry out more than 40 cycles in order to prevent the formation of nonspecific products.
Step 2: Denaturation step
95ÂşC for 30 s or 97ÂşC for 15 s
For complex templates (genomic DNA) begin with denaturation for 5-10 min before the actual cycles (Hot Start)
G/C content greater than 50% increases the denaturation temperature
T1/2 = half life of Taq at specific temperature<
Inefficient denaturation is a frequent cause of errors. However:
T1/2 Taq at 92.5ÂşC = 2 hours
T1/2 Taq at 95.0ÂşC = 40 min
T1/2 Taq at 97.5ÂşC = 5 min
T1/2 Taq at 97.5ÂşC + 10% glycerin = 23 min
T1/2 Pwo at 100ÂşC = 2 hours
Step 3: Annealing step
For standard primers
(approx. 20 nucleotides [nt]; 1 ÂµM; 100% match)
Approx. 20 s
Tm = melting point of DNA
Ta = annealing temperature
Low concentrations and long primers extend necessary annealing times. The annealing temperature (Ta) can be estimated on the basis of the melting temperature (Tm) using the following formulae:
Tm = (A+T) x 2 + (C+G) x 4 [Wallace], up to ca. 20 nt
Tm = 81.5ÂşC + 16.6 (log[Na+]) + 0.41 (%G+C) â€“ (500/n) â€“ 0.61 (%FA)
[Meinkoth and Wahl]; FA = Formamide
Ta = Tm â€“ (5 to 10ÂşC)
Step 4: Elongation step
Approx. 60 bp are synthesized every second under optimum conditions
2 Kbp require approx. 1 min.
The shorter the fragment, the easier it is to control the reaction
200â€“500 bp fragments are sufficient for most detection reactions
PCR Tips, tricks, techniques and calculations ~ Reagents
This table provides newcomers to the PCR with an overview of the common rules and concentrations which should be observed when designing new experimental setups.
There are, of course, a number of exceptions and special points which, due to lack of space, cannot be covered here.
References: Sambrook, J., Fritsch, E. F., Maniatis, T.; (1989) Molecular cloning, 2nd ed., Cold Spring Harbor Laboratory Press.
The most common components
(as 10-x buffers):
500 mM KCl
100 mM Tris-HCl, pH 8.3 (at 25ÂşC) or 150â€“200 mM (NH4)2SO4 with 500â€“750 mM Tris-HCl, pH 9 (at 25ÂşC)
1â€“2% TritonÂ® X-100 or 0.1% TweenÂ®
10â€“15 mM MgCl2 (usually available separately)
The buffers delivered with the polymerase are specially tailored to this variety and it is often not possible to use them with enzymes from other manufacturers.
NaCl concentrations greater than 50 mM inhibit the Taq Polymerase.
MgCl2 must be added (if not present in buffer) as Mg2+ is essential as a co-factor for the DNA polymerase.
0.5â€“3.5 mM in the assay
Essential as a co-factor of the DNA polymerase
If dUTP is used instead of dTTP, the Mg2+ concentration usually must be increased (max. 5 mM).
Concentration too high: promotes the amplification of nonspecific fragments (smears appear); increases the melting temperature.
Concentration too low: reduces annealing efficiency and the synthesis rate. polymerase
1 unit / 50 Âµl reaction sample
Concentration too high: reduced specificity
Concentration too low: reduced efficiency
Please see point 2, "Denaturation stepâ€ť!
Storage in 10 mM, pH 7.0 aliquots
20â€“200 ÂµM in the assay
Concentration too high: leads to mispriming and the misincorporation of nucleotides.
Following a successful PCR, theoretically the major part of the dNTPs are left over.
All nucleotides must have the same concentration.
Modified nucleotides must have a higher concentration.
0.1â€“1 ÂµM in the assay
Length: approx. 15â€“30 nt
Secondary structures should not be able to form (Stemloop, Hairpin).
Primer must not be complementary at the 3' ends as this will lead to the formation of primer dimers.
Both primers should have the same Tm
No stretches of individual nucleotides.
G or C at the 3' end improves binding.
TritonÂ® is a registered trademark of Union Carbide Chemicals and Plastics Co., Inc. TweenÂ® is a registered trademark of ICI Americas, Inc.
PCR Tips, tricks, techniques and calculations ~ Detection of nucleic acids and proteins
Recommended Agarose Gel Percentages for Resolution of DNA
DNA size range
Recommended Polyacrylamide Gel Percentages for Resolution of DNA
DNA size range
Recommended Polyacrylamide Gel Percentages for Resolution of Proteins
protein size range
Dye Migration in Polyacrylamide Non-Denaturing Gels
Dye Migration in Polyacrylamide Denaturing Gels
Physical Properties of Nucleotides
max (pH 7.0)
Absorbance at max1 M solution (pH 7.0)
Biophysical Data for Deoxynucleoside Triphosphates
Molecular Weight (Daltons)
Molar Extinction Coefficient and Peak Absorbance Wavelength (pH 7.0)
15,200 at 259 nm
9,300 at 271 nm
13,700 at 253 nm
9,600 at 267 nm
Conversion to Weight (Âµg/ml)
Double-stranded DNA (dsDNA):
A260 = OD260 = 1 for a 50 Âµg/ml solution
Single-stranded RNA (ssRNA):
A260 = OD260 = 1 for a 40 Âµg/ml solution
Single-stranded DNA (ssDNA):
A260 = OD260 = 1 for a 33 Âµg/ml solution
Molar Extinction Coefficient x Concentration x Pathlength
(usually cuvette width)
PCR Tips, tricks, techniques and calculations ~ Calculating primer quantity
PCR Tips, tricks, techniques and calculations ~ Genetic code, properties and molecular structure of amino acids
Nomenclature and properties of amino acids
Major properties of side chains
No side chain
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updated December 10th,2015
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