IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-1

=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)
4 µl      10*H buffer 1 µl      EcoRI 1 µl      PstI 20 µl     Plasmid 14 µl     dH20 -- 40 µl     Total
 * purpose:use pSB1A2 as vector.Should knock out fragment E0040 first.
 * use Double digesting test for gel extraction.
 * Digestion system contains:

electrophoresis result before gel extraction

 * from left to right:
 * 1) pSB1A2-1 @ EcoRI/PstI
 * 2) pSB1A2-2 @ EcoRI/PstI
 * 3) marker

electrophoresis result after gel extraction

 * from left to right:
 * 1) pSB1A2(without E0040)
 * 2) 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)

 * 1) 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℃.
 * 2) Monitor OD changes after shaking for 90 min.
 * 3) Harvest at 120 min (with OD 0.50) by cooling down the bacteria.
 * 4) Transfer each strain to a centrifugal tube, and balance it using another tube.
 * 5) Cool down strains to zero centigrade (Leave the culture on ice for 15 min)
 * 6) Meanwhile, cool down centrifuge to 4℃, and put 100 mM CaCl2 and 15% glycerol with 100 mM CaCl2 on ice.
 * 7) After cooling down the bacteria, centrifugate at 3500 rpm, +4℃ for 10 min. Throw away the cleared liquid.
 * 8) Gently resuspend pellets in half culture volume (25 mL) sterile and cold 100 mM CaCl2
 * 9) Leave the suspension on ice for 20 min
 * 10) Centrifugate at 3500 rpm, +4℃ for 10 min. Throw away the cleared liquid
 * 11) Gently resuspend pellets in 1/20 culture volume (2.5 mL) 15% glycerol with CaCl2
 * 12) Separate into EP tubes with 100 μL, cool them down thoroughly in liquid nitrogen, then hold in -80℃ overnight before using them.