Team:XJTLU-CHINA/Protocols PCR
PCR using Q5 Hi-Fi DNA polymerase Master Mix
Reaction setup:
The template DNA should keep an amount of 1-1000 ng
Thermocycler conditions
Clarification:
- The sequences in red are where the primers anneal with the biobrick affixes. And the 5’overhangs side with the sequences in red are designed for Gibson Assembly with linearized pNZ8148 digested by PstI due to a 21-nucleotide overlap between them.
- The reason of setting two different cycles is that, at the beginning of PCR, the annealed parts between primers and templates are only sequences in red. As PCR proceeds, 5’overhangs are added to the entire genes, so the rest reaction of PCR becomes the one between the whole primers and the whole genes, because of which the Tm varies compared to the former.
Applications in this project: Amplification of BBa_K2309028, BBa_K2309003 and BBa_K2309004
Collaborators and Supporters
