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Revision as of 06:27, 17 October 2018

Notebook

Basic techniques in Molecular biology

Polymerase chain reaction

Total volume50/μL
DDW36
10×Buffer5
dNTP（2.5mM）5
FP（10μM）1
RP（10μM）1
DNA template1
EasyPfu DNA Polymerase1

Thermocycling

The PCRmachine should be set to run the following steps

Pre-denaturation94℃5min
transsexual94℃30s30circles
annealingTm-5℃30s
extend72℃0.5kb/min
extend72℃5min

extraction plasmid

1-4 mL of the bacterial solution is centrifuged at 12,000 rpm for 2 min, and the bacterial solution is discarded.

2) Resuspend by adding 200μL SolutionI, then add 400μL SolutionII and invert upside and down slowly until the bacterial solution is transparent. Next add 300μL SolutionIII and mix. Further, 300 ul of chloroform is added and mixed, and centrifuge at 13,000 rpm for 2 minutes. 700 ul of the supernatant is removed into a new 1.5 mL tube, and add 490 μL of 70% isopropanol.Next centrifuge at 13,000 rpm for 2 minutes, and the supernatant is discarded. After rinsing the precipitate twice with 500 μL of 75% ethanol, centrifuges at 12,000 rpm for 2 min.Finally,dry at 65℃ and dissolve with 15-20 mL of sterile water previously heated.

PCR Purfication and gel extraction

PCR Purfication