Reactions – PCR – first amplification


1. 12.8 μl ddH2O
2. 1.8 μl 10mM MgCl2
3. 2.0 μl 10mM dNTPs
4. 2.5 μl 10x Pfu or KlenTaqLA PCR Buffer (both include 15 mM MgCl2)
5. 2.5 μl 2μM primer1
6. 2.5 μl 2μM primer2
7. 0.03 μl of 10 U KlenTaqLA (1 U/1 kb amplified in 100 μl PCR reaction)

Mix and add 24 μl of master mix to each reaction. Add 1 μl DNA (5-100 ng/μl).

1. 68°C for 60 sec. (prewarm)
2. 93°C for 5 sec. (denature)
3. 52°C for 35 sec. (anneal)
4. 68°C for 90 sec. (extend) – time depends product length (at least 30 sec / 1 kb)
5. 68°C for 7 min. (final extend)

Repeat the parameters 2 – 4 30 times. I have found that a 2 or 5 minute “hot start” at 94°C does not work that well, but the 68°C preheating ensures that the first 93°C denaturing step will denature completely.
There is no need to test the quality of the PCR product after the first reaction.

Reactions – PCR – second (nested) amplification

1. 7.6 μl ddH2O
2. 1.2 μl 25mM MgCl2
3. 1.2 μl 10mM dNTPs
4. 1.5 μl 10x (NH4)2SO4 PCR Buffer (no MgCl2)
5. 1.5 μl 2μM primer1
6. 1.5 μl 2μM primer2
7. 0.3 μl of 1 U Taq polymerase

Mix and add 14 μl of master mix to each reaction. Add 1 μl PCR product from previous reaction. Depending on the primer, the PCR product may need to be diluted (1:5 to 1:50).

1. 68°C for 60 sec. (prewarm)
2. 93°C for 5 sec. (denature)
3. 55°C for 35 sec. (anneal)
4. 68°C for 90 sec. (extend) – time depends product length (at least 30 sec / 1 kb)
5. 68°C for 7 min. (final extend)

The nested design can be modified to a half-nested approach. The half-nested approach uses only one nested primer while the other primer is the same as in the original amplification reaction. Unless the primers are very degenerate or have too many multiple binding sites, this approach also works well.

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