IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-14
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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.
- PCR program condition 1: 94℃ 5min, 94℃ 30s, 51℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end.
- 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.