IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-19: Difference between revisions
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===electrophoresis result === | ===electrophoresis result === | ||
==colony PCR Test for R751, pSC101== | ==colony PCR Test for R751, pSC101(III)== | ||
*Test Lb- R751 plate, Lb- pSC101 have the correct plasmid. | *Test Lb- R751 plate, Lb- pSC101 have the correct plasmid. | ||
*Test plate:LB- R751, Lb- pSB101, Tc+ R751-pSC101, Tc+ Dh5α-pSC101, Tc+ R751-pSC101, Amp+ Dh5α-R0040 | *Test plate:LB- R751, Lb- pSB101, Tc+ R751-pSC101, Tc+ Dh5α-pSC101, Tc+ R751-pSC101, Amp+ Dh5α-R0040 |
Revision as of 23:54, 19 August 2007
Tandem Ori-T by Qu Mingzhi, Ren Ze
mini-prep R-OriT, S-Orit (pSB1A2)
- using Transgen mini plasmid puriflication kit.
- 50µL after purflication
mini-prep double digesting test
- R-OriT, S-OriT digestion system contains(Standard Assembly fragment):
4 µl 10*H buffer 1 µl EcoRI 1 µl PstI 20 µl Plasmid 14 µl dH20 -------------------------- 40 µl Total
electrophoresis result
colony PCR Test for R751, pSC101(III)
- Test Lb- R751 plate, Lb- pSC101 have the correct plasmid.
- Test plate:LB- R751, Lb- pSB101, Tc+ R751-pSC101, Tc+ Dh5α-pSC101, Tc+ R751-pSC101, Amp+ Dh5α-R0040
- primer :R751 OriT primer, pSC101 primer.
- according to <Colony PCR STANDARD PROTOCOL>
- PCR system contains(each well):
0.5 µl Primer 1(100uM) 0.5 µl Primer 2 2 µl dNTP(2.5uM) 2.5 µl 10X Taq Buffer 0.25 µl Taq 19 µl dH20 1 µl template -------------------------- ~25 µl Total
- PCR program condition 1: 94℃ 5min, 94℃ 30s, 53℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end.
- PCR program condition 2: 94℃ 5min, 94℃ 30s, 57℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end .
electrophorsis result
- top:from left to right
- 1-4 pSC101
- 5-8 R751
- 9-16 R751-pSC101
- 17 Maker(DL2000 plus)
- botton:from left to right
- 18-21 Dh5α-pSC101
- 22-23 Dh5α-R0040