IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-14

Tandem OriT by Qu Mingzhi

PCR P-OriT(pSC101) & R-OriT(R751)

• get P-OriT primer, R-OriAT from the fridge, stored as ~100uM.

PCR system contains(each well):

0.5  µl      Primer 1
0.5  µl      Primer 2
2    µl      dNTP
2.5  µl      10X Taq Buffer
0.25 µl      Taq
19   µl      dH20
1    µl      OriT template
--------------------------
~25  µl      Total

• use different PCR program to test efficiency.

1. PCR program condition 1: 94℃ 5min, 94℃ 30s, 51℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end.
2. PCR program condition 2: 94℃ 5min, 94℃ 30s, 55℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end .

Primer final concentration 1uM.

electrophorsis result

• from left to right:
  • 1-8 pSC101 OriT
  • 9-12 R751 OriT

OriT PCR product purification

• from left to right:
  • 1-4 pSC101 OriT after gel purification
  • 6-6 r751 OriT after gel purification

transformation E0240

• get E0240 from biobrick.
• NEXT DAY: received 50+ clones.