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