IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-1
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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.
electrophoresis result
Lock & Key by Yu tao
oriT Knock Out
- By Xu Anting
Competent Cell preparation (End)
- After re-activation in LB liquid, Add 500 μL→50 mL (1:100) with LB- in a 250 mL flask, and incubate under shaking in 37℃.
- Monitor OD changes after shaking for 90 min.
- Harvest at 120 min (with OD 0.50) by cooling down the bacteria.
- Transfer each strain to a centrifugal tube, and balance it using another tube.
- Cool down strains to zero centigrade (Leave the culture on ice for 15 min)
- Meanwhile, cool down centrifuge to 4℃, and put 100 mM CaCl2 and 15% glycerol with 100 mM CaCl2 on ice.
- After cooling down the bacteria, centrifugate at 3500 rpm, +4℃ for 10 min. Throw away the cleared liquid.
- Gently resuspend pellets in half culture volume (25 mL) sterile and cold 100 mM CaCl2
- Leave the suspension on ice for 20 min
- Centrifugate at 3500 rpm, +4℃ for 10 min. Throw away the cleared liquid
- Gently resuspend pellets in 1/20 culture volume (2.5 mL) 15% glycerol with CaCl2
- Separate into EP tubes with 100 μL, cool them down thoroughly in liquid nitrogen, then hold in -80℃ overnight before using them.