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

Tandem OriT by Qu Mingzhi

Ligation: PlacI-I741051 <- E0240 & R0010 <- E0240 (III)

• 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:

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

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

• p1,p2,p3 primer: stored as 100uM.
• Use both taq and pfu.
• PCR system contains:

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

• Primer final concentration 1uM.
• Add a drop of liquid paraffin to each system.
• Try 4 anneal temperature: 51℃, 54℃, 57℃, 60℃.
• PCR program setting:

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

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.