Protocol for Screening Bacterial Colonies by H2O Lysis
PCR
1) Prepare fresh LB/Agar/antibiotic plates by dividing them into squares about
1cmx1cm.
2) Setup as many PCR tubes as necessary and add 20ul of water to each (autoclaved
milli-Q or ultrapure).
3) Pick colonies by first spotting onto one grid division, and then into a watercontaining tube. Repeat until the desired number of colonies have been picked.
4) Keep tubes on ice while making the PCR mix as follows:
462.5ul water
125ul 10X buffer
50ul forward primer (50ng/ul)
50ul reverse primer (50ng/ul)
25ul dNTPs (10mM)
25ul MgCl2 (50mM)
12.5ul Taq Polymerase (5U/ul)
750ul total Good for 25 reactions
5) Add 30ul of the above reaction mix to each tube and cap.
6) Prepare positive control containing 0.5ng of plasmid DNA. Also include negative
controls with no template DNA, only the above reaction mix.
7) Run PCR program.