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 5. Run 30 to 35 cycles in a thermocycler using the following PCR program (see Hint #1
