IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-31: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Qu Mingzhi (talk | contribs) (New page: ==Tandem OriT== *by Qu Mingzhi) |
No edit summary |
||
(13 intermediate revisions by 2 users not shown) | |||
Line 1: | Line 1: | ||
== | =Tandem OriT By Qu Mingzhi= | ||
* | ==PCR OriT== | ||
*get F-OriT primer, primer vf,vf2,vr,pf1,pr1 stored as ~100uM. | |||
*PCR system contains (totally 50uL):1uL Primer pf1, 1uL Primer pr1, 4uL dNTP, 0.5uL Taq, 1uL OriT template, 37.5uL dH20, 5uL 10X buffer, | |||
*use different PCR program to test efficiency. | |||
#PCR program condition 1: 94℃ 5min, 94℃ 30s, 51℃ 30s, 72℃ 30s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end. | |||
#PCR program condition 2: 94℃ 5min, 94℃ 30s, 55℃ 30s, 72℃ 30s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end . | |||
*Primer final concentration 1uM. | |||
===electrophorsis result=== | |||
*from ''left'' to ''right'': | |||
#37℃ @ program 1 | |||
#30℃ @ program 1 | |||
#37℃ @ program 2 | |||
#37℃ @ program 2 | |||
#marker | |||
[[Image:Peking_2007-7-31_PCR_OriT_T51_37_30_T55_37_30(2).jpg]] | |||
==OriT PCR product purification== | |||
*use Transgen kit. | |||
*40uL after purflication | |||
===electrophorsis result=== | |||
*from ''left'' to ''right'': | |||
#37℃ @ program 1 before purflication | |||
#30℃ @ program 2 before purflication | |||
#30℃ @ program 1 after purflication | |||
#37℃ @ program 2 after purflication | |||
#marker | |||
*the '''left two channels''' are used to re-test the aboabnormal line that showned on PCR product electrophorsis test. | |||
[[Image:Peking_2007-7-31_PCR_purflication_oriT.jpg]] | |||
==transformation OriT== | |||
*Use pEASY-3 as cloning vector .(Amp+) | |||
*tramsformation system contains :3uL PCR product(after purflication), 1uL pEasy-T | |||
*the Culture Dish(LB/Amp+) are trated with 5uL 1M IPTG & 40uL 40mg/mL x-Gal. | |||
*'''NEXT DAY:''' 10 white cloney received. | |||
=Lock & Key By Yu Tao= | |||
=oriT Knock Out= | |||
*By Xu Anting | |||
==Competent Cell preparation== | |||
*After activation of three strains (F/R/S) plates, shake them with LB liquid (pSC101 in Tc+) in 37℃ for 16 hours. |
Latest revision as of 06:39, 24 August 2007
Tandem OriT By Qu Mingzhi
PCR OriT
- get F-OriT primer, primer vf,vf2,vr,pf1,pr1 stored as ~100uM.
- PCR system contains (totally 50uL):1uL Primer pf1, 1uL Primer pr1, 4uL dNTP, 0.5uL Taq, 1uL OriT template, 37.5uL dH20, 5uL 10X buffer,
- use different PCR program to test efficiency.
- PCR program condition 1: 94℃ 5min, 94℃ 30s, 51℃ 30s, 72℃ 30s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end.
- PCR program condition 2: 94℃ 5min, 94℃ 30s, 55℃ 30s, 72℃ 30s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end .
- Primer final concentration 1uM.
electrophorsis result
- from left to right:
- 37℃ @ program 1
- 30℃ @ program 1
- 37℃ @ program 2
- 37℃ @ program 2
- marker
OriT PCR product purification
- use Transgen kit.
- 40uL after purflication
electrophorsis result
- from left to right:
- 37℃ @ program 1 before purflication
- 30℃ @ program 2 before purflication
- 30℃ @ program 1 after purflication
- 37℃ @ program 2 after purflication
- marker
- the left two channels are used to re-test the aboabnormal line that showned on PCR product electrophorsis test.
transformation OriT
- Use pEASY-3 as cloning vector .(Amp+)
- tramsformation system contains :3uL PCR product(after purflication), 1uL pEasy-T
- the Culture Dish(LB/Amp+) are trated with 5uL 1M IPTG & 40uL 40mg/mL x-Gal.
- NEXT DAY: 10 white cloney received.
Lock & Key By Yu Tao
oriT Knock Out
- By Xu Anting
Competent Cell preparation
- After activation of three strains (F/R/S) plates, shake them with LB liquid (pSC101 in Tc+) in 37℃ for 16 hours.