Protocol
1. PCR system
1) 20μL system is used for verification
Taq:
Super mix:
2) 50μL system for PCR target genes
Pfu mix:
DNA: plasmid 50ng; genome 100~200ng
3) 20μL PCR system for microbes
Super mix:
Bacteria (after picking on the board, back up on the new board and rinse it in the system that has been prepared)
4) Fusion PCR
1. Basic PCR2. Use the PCR product of step 1 as template to do PCR3. Use the PCR product of step 2 as template to do PCR, but first five cycles don't add primer, add primer at the sixth cycle and continue PCR.
The system of step 2: H2O 21μL 2x primeSTAR 25μL R+F-Primer 2 μL Template1 1μL Template2 1μL
2. Plasmid concentration
1. Dilute collected DNA to 400μl. (If the plasmid is eluted by ddH2O, then it is diluted by ddH2O as well; If the plasmid is eluted by elution buffer, then it is diluted by elution buffer as well)