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.

4 µl       10*H buffer
1 µl       EcoRI
1 µl       PstI
20 µl      Plasmid
14 µl      dH20
--------------------------
40 µl      Total

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.

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.