Annealing Oligos for Cloning

  A. Reaction Components:

2 ul 10X PCR buffer w/ 15 mM MgCl2

1. Design oligos (the Oligator is a useful tool for this)
2. Order oligos at 100 uM concentration (eg. IDT)

ul 2 mM dNTPs (working concentration 2 mM each) 

3 1 ul forward primer (20 pmol)

1 ul reverse primer (20 pmol)

1 ul Taq DNA polymerase

12 ul dH2O

19 ul total

add 1 ul template DNA

Create master mix if desired

  B. Thermal Profile

1 circle of : 94 C 4 mins

30 circles of: 94 C 15 sec.; annealing temp. 15 sec.; 74 C 30 sec.

1 circle of : 74 C 4 mins

annealing temp. should be about 4 C below the lower of the Tm values for the two primers.