PCR approach in 20 μL with hot-start (qualitative PCR)

Materials

The same as for PCR approach in 50 µL

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	12 µL
☐	10x Taq/Pfu buffer	1.5 μL
☐	Primer 1	0.4 μL (10 pmol)
☐	Primer 2	0.4 μL (10 pmol)
☐	Template	0.2 μL (~10 ng)
☐	dNTPs	0.2 μL
☐	Total	15 μL

✓ Enzyme Mix:

☐	H2O	4 µL
☐	10x Taq/Pfu buffer	0.5 μL
☐	Taq Polymerase	0.5 μL
☐	–	–
☐	–	–
☐	–	–
☐	Total	5 μL

In contrast to preparative PCR, only a single temperature is used for hybridization and the number of cycles is reduced to 20, as the signal of the amplified sequence is then already strong enough to be detected via ethidium bromide staining.

1	Hot start	94°C
Hold
2	Denauturation	94°C	20 seconds
3	Hybridization	55°C	20 seconds
4	Elongation	72°C	60 seconds per 1k bp
30 repetitions of steps 2-4
5	Elongation	72°C	5-10 minutes
6	Stop	8°C