PCR from Plasmid and Genomic DNA
Must use high fiderity polymerase such as Phusion DNA Polymerase
Genomic DNA
[edit]25 ~35 cycles
25 or 50 ul reaction
5 ~10 ul genomic DNA, at least 100 copies
0.5~1 ul F 10 uM primer
0.5~1 ul R 10 uM primer
0.5~1 ul 10 mM dNTP
5 or 10 ul 5X Phusion or Q5 Buffer
0.5 or 1 ul Phusion
Run 15 ul + 5 ul 6X GRGreen Loading Buffer on gel.
Clone PCR product using blunt-end cloning protocol
Plasmid DNA
[edit]25 cycles
25 or 50 ul reaction
0.5 ul plasmid DNA, at least 100 copies
0.5~1 ul F 10 uM primer
0.5~1 ul R 10 uM primer
0.5~1 ul 10 mM dNTP
5 or 10 ul 5X Phusion or Q5 Buffer
0.5 or 1 ul Phusion
Run 15 ul + 5 ul 5X GRGreen Loading Buffer on gel.
Clone PCR product using blunt-end cloning protocol
PCR Positive Control
Usually you need to run at least one positive control --> your PCR is working
and one negative control.-->identify false positive