PCR approach in 50 μL with hot-start and/or touch-down (preparative PCR)

Materials

20 mM dNTPs
10x Taq/Pfu buffer
2 Primers 10 μM
DNA template, 100 – 200 ng/μL
DNA polymerases (Pfu, Taq)

Procedure

As usually more than one PCR sample is run simultaneously, reaction and enzyme mix are prepared separately. The enzyme mix is added to the reaction mix after starting the PCR and after pre-heating the reaction mix to 94 ºC (hot start). A typical 50 μL approach that yields enough material for later cloning procedures is made up as follows:

✓ Reaction Mix:

☐	H2O	32 µL
☐	10x Taq/Pfu buffer	4 μL
☐	Primer 1	1 μL (10 pmol)
☐	Primer 2	1 μL (10 pmol)
☐	Template	1 μL (~10 ng)
☐	dNTPs	1 μL
☐	Total	40 μL

✓ Enzyme Mix:

☐	H2O	7.5 µL
☐	10x Taq/Pfu buffer	1 μL
☐	Pfu Polymerase	0.5 μL
☐	Taq Polymerase	1 μL
☐	–	–
☐	–	–
☐	Total	10 μL

Touch-down PCR:

1	Hot start	94°C	Pause – addition of enzyme mix
7 repetitions of steps 2-4 with hybridization temperature - 1 ºC per cycle (touch-down)
2	Denauturation	94°C	20 seconds
3	Hybridization	64-58°C	20 seconds
4	Elongation	72°C	depending on fragment size (1 min/750 bp)
30 repetitions of steps 2-4 with hybridization temperature of 58 ºC
5	Elongation	72°C	5-10 minutes
6	Stop	8°C

(An alternative to touch-down PCR is a gradient PCR, with a gradient of annealing temperatures usually between 50 – 64°C)