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)2. Add 900μl absolute ethanol to precipitate DNA, and 40μl NaAc (3M PH=5.2) to help precipitation. Be sure to mix it up.3. Put the reactant in ice-bath for 1 hour, or put it under -20 degrees Celsius overnight.
4. Centrifuge the reactant under 12000rpm for 10 minutes, then throw away the supernatant.5. Add 1ml 75% ethanol (the 75% ethanol is prepared by using absolute ethanol rather than ethanol for disinfection) and mix up to wash away salt ions.
6. Centrifuge the reactant under 12000rpm for 5 minutes, then throw away the supernatant.7. Centrifuge the reactant for another 3-4 seconds and use transfer liquid gun to throw away residual supernatant.
8. Centrifuge the reactant for another 3-4 seconds and use transfer liquid gun to throw away residual supernatant.
9. Open the centrifuge tube and expose the reactant to air. Put the centrifuge tube into incubator at 37 degrees Celsius for 15 minutes until the plasmid turn into transparent.
10. Add appropriate volume of ddH2O to dilute the plasmid according to precipitation capacity.
11. Use 1μl solution and add 9μl ddH2O to dilute it, then use electrophoresis to measure its quantity.