Colony PCR to check for insert

Polymerase Chain Reaction (PCR)
5 ul 10X PCR buffer (contains buffering agent, KCl, 15mM MgCl2)
4 ul dNTPs, stock is 2.5 mM (each nucleotide is at 2.5 mM)
2 ul 5’ primer, 10 pmol/ul stock
2 ul 3’ primer, 10 pmol/ul stock
water (Millipore filtered), q.s. to 50 ul (quantity sufficient to give 50
1.0 ul taq polymerase
1-5 ul DNA template (about 100-500 ng)
50 ul total volume
a. make a cocktail for the number of reactions plus 1 extra. Make sure
to include a neg. control reaction that lacks template. Dole out
cocktail mixture and then add template.
b. can cut recipe in half (25 ul total volume reactions)
c. alternatively, use a 2X Taq polymerase mixture that contains
polymerase, buffer, salts and dNTPs; just add primers, template and
d. When screening bacterial colonies for recombinant plasmids, pick a
portion of a colony using a pipet tip or sterile toothpick and swirl into
reaction in place of template. Include a neg. control colony reaction.
-Typical PCR conditions:
1. 94 C for 5 minutes
2. 94 C for 30 seconds
3. 55 C for 30 seconds (temp. depends on Tm of primers; want ~5°
below lowest primer Tm)
4. 72 C for 1’ (1 minute per kb of target DNA)
5. -repeat back to step 2, 29-39 times depending on target template
conc. (34 is typical).
6. 72 C for 10 minutes to finish DNA ends
7. hold at 4 C (so you can go home without worry)
8. store at -20 C until further processing
9. add Loading buffer to 1-2X. Gel check 5-10 ul.