IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-21

=Tandem OriT by Mingzhi Qu=

Ligation: PlacI-I741051 <- E0240 & R0010 <- E0240 (III)
2  µl     vector 3  µl     E0240 fragment 0.5 µl    super fast T4 DNA ligase 1  µl     10X T4 ligase buffer 3.5 µl    dH20 -- 10 µl    Total
 * Ligate the E0240 fragment and PlacI-I741051 vector.
 * Ligate the E0240 fragment and R0010 vector.
 * use super fast T4 DNA ligase
 * super fast T4 DNA ligase Ligation system contains:

transformation PlacI-I741051 <- E0240 & R0010 <- E0240(III)
NEXT DAY: Received clones.

=Lock & Key by Yu Tao=

Colony PCR: J01008->R0010

 * Select 18 clones on the J01008->R0010 plates for colony PCR.
 * Target: positive J01008->R0010 clones.
 * Use vf2 and vr primers.
 * Set 2 anneal temperature: 60℃ and 62℃.

Electrophorsis Result

 * from left to right:
 * 1) $$$ @ EcoRI/PstI
 * 2) marker (DL2000 Plus)

PCR: J01008
for taq:                       for pfu: 1 µL     Primer p1             1 µL      Primer p1 1 µL      Primer p2             1 µL      Primer p2 1 µL      Primer p3             1 µL      Primer p3 4 µL      dNTP                  5 µL      dNTP 0.5 µL   Taq                   1 µL      Pfu 5 µL     10 X buffer           5 µL      10 X buffer 37.5 µL  ddH20                 37.5 µL   ddH20 1 µL     0.1M MgSO4 -- 50 µl     Total Step1    94℃ 5min Step2    94℃ 30s Step3    51℃/54℃/57℃/60℃ 30s Step4    72℃ 30s Step5    Go to step 2 for 4 more times Step6    72℃ 10min End
 * p1,p2,p3 primer: stored as 100uM.
 * Use both taq and pfu.
 * PCR system contains:
 * Primer final concentration 1uM.
 * Add a drop of liquid paraffin to each system.
 * Try 4 anneal temperature: 51℃, 54℃, 57℃, 60℃.
 * PCR program setting:

The above PCR Product Purification

 * Use Transgen EasyPure PCR Purification Kit.
 * 50 uL per tube after purification.

Double Digestion: the above PCR product

 * Digesting all the PCR product with XbaI/PstI.
 * Each digestion system contains:

4 µl      10*M 1 µl      XbaI 1 µl      PstI 10 µl     Plasmid 20 µl     ddH20 4 µl      BSA -- 40 µl     Total


 * 37℃ overnight.