What is asymmetric PCR? A PCR in which the predominant product is a single-stranded DNA, as a result of unequal primer concentrations. As asymmetric PCR proceeds, the lower concentration primer is quantitatively incorporated into double-stranded DNA. The higher concentration primer continues to primer synthesis, but only of its strand.
Asymmetric PCR Protocol
1. Pick a phage plaque and place in 100 ul TE or scrape a fresh colony of a bacterial transformant of choice and place in 50 ul of TE/TX100 in a microcentrifuge tube.
2. Heat the tube for 10 min at 95C.
3. Centrifuge at maximum speed for several minutes in a microcentrifuge to pellet cell debris. Collect the supernatant.
4. Add the following components in a PCR tube:
5 ul of phage or bacterial extract (from Step #A3)
50 uM of dNTPs
50 pmol of Primer 1
1 pmol of Primer 2
in 1X PCR Reaction Buffer to give a final reaction volume of 50 to 100 ul
2.5 Units of Taq polymerase