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.
Lock & Key By Yu Tao
Ligation and Transformation: T3-J01010 and T3-J01008
Transformation Result
- Proper clones grow on the plate of T3-J01010.
- No single clone grow on the plate of T3-J01008, probably because the used competent cells were Competent Cells II.
- I suggest we need to religate and transform the T3-J01008.
Mini-prep: T-J01010, T-J01008 and Competent Cell II negative control clones
- Using Transgen mini plasmid purification kit.
- 50 uL per tube after purification, 2 tubes per type of plasmids.
Mini-prep Double Digesting Test Result
- Digesting all the plasmids above with EcoRI/PstI.
- Each digestion system contains:
1 µl 10*H buffer 0.25 µl EcoRI 0.25 µl PstI 5 µl Plasmid 3.5 µl ddH20 -------------------------- 10 µl Total
- 37℃ culutre overnight.
- from left to right:
- T-J01010-1 @ EcoRI/PstI
- T-J01010-2 @ EcoRI/PstI
- T-J01008-1 @ EcoRI/PstI
- T-J01008-2 @ EcoRI/PstI
- Competent Cells II @ EcoRI/PstI
- marker (DL2000 Plus)
- Conclusion: Obviously, only the T-J01010 seems correct.
Double Digestion: R0040<-J01010 and R0010<-J01008
- Digesting R0040<-J01010 with EcoRI/SpeI and R0010<-J01008 with SpeI/PstI.
- Each digestion system contains:
4 µl 10*H 1 µl SpeI 1 µl EcoRI/PstI 20 µl Plasmid 14 µl ddH20 -------------------------- 40 µl Total
- 37℃ overnight.