IGEM:PKU Beijing/2009/Notebook/AND Gate 1/Output/2009/06/27/Zhangs

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Miniprep CI plasmid
Some improvements:
1.the EB be put in 65°C water bathing before elution;
2.50 uL EB to elute;
3.after PW liquid, centrifuge 10 min before the next step;
4.the rpm increased to 13000 per min
A260/A280 = 1.92 A260 = 0.120 CI = 300ng.uL

Previous double digestion protocol seemed to be lack of efficiency, so we tried new protocol today.
New double digestion system:
For front insert:

10μL plasmid
2μL 10×H beffer
1.5μL EcoRI
1.5μL SpeI
5μL ddH2O
20μL Total

For front vector:

10μL plasmid
2μL 10×M beffer
1.5μL EcoRI
1.5μL XbaI
5μL ddH2O
50μL Total

Electrophoresis to test
Samples: 10μL digestion system+2μL DNA Dye
Control: 5μL plasmid+2μL DNA Dye
Marker: 10μL DL2000 plus+2μL DNA Dye
React for 10 minutes
Result (from left to right):
Marker, CI control, CI, 1-23L control, 1-23L, 1-4H control, 1-4H
PKU 20090627 Zhangs 1.JPG

Gel extraction Extract CI insert, 1-4H vector and 1-23L vector