IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-1: Difference between revisions
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=Tandem Ori-T by Qu Mingzhi= | |||
==Amplification Culture of Ori-T== | ==Amplification Culture of Ori-T== | ||
select Positive OriT-pEASY-3 Colonies from Plate,Culture in liquid LB,waiting for mini-prep. | select Positive OriT-pEASY-3 Colonies from Plate,Culture in liquid LB,waiting for mini-prep. | ||
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*PCR program condition :94℃ 5min, 94℃ 30s, 60℃ 30s, 72℃ 30s, Go to step 2 for 5 cycles, 72℃ 10min, 4℃ end. | *PCR program condition :94℃ 5min, 94℃ 30s, 60℃ 30s, 72℃ 30s, Go to step 2 for 5 cycles, 72℃ 10min, 4℃ end. | ||
===electrophoresis result=== | ===electrophoresis result=== | ||
=Lock & Key by Yu tao= | |||
Revision as of 04:52, 9 August 2007
Tandem Ori-T by Qu Mingzhi
Amplification Culture of Ori-T
select Positive OriT-pEASY-3 Colonies from Plate,Culture in liquid LB,waiting for mini-prep.
Double digesting test for pSB1A2(include E0040)
- purpose:use pSB1A2 as vector.Should knock out fragment E0040 first.
- use Double digesting test for gel extraction.
- Digestion system contains:
4 µl 10*H buffer 1 µl EcoRI 1 µl PstI 20 µl Plasmid 14 µl dH20 -------------------------- 40 µl Total
electrophoresis result before gel extraction
- from left to right:
- pSB1A2-1 @ EcoRI/PstI
- pSB1A2-2 @ EcoRI/PstI
- marker
electrophoresis result after gel extraction
- from left to right:
- pSB1A2(without E0040)
- marker
PCR crRNA
- PCR system contains (totally 50uL):0.5uL Primer 1(50uM), 0.5uL Primer 2(50uM), 4uL dNTP(2.5mM), 0.5uL Taq(5u/uL), 1uL OriT template, 39.5uL dH20, 5uL 10X buffer.
- PCR program condition :94℃ 5min, 94℃ 30s, 60℃ 30s, 72℃ 30s, Go to step 2 for 5 cycles, 72℃ 10min, 4℃ end.
