IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-4
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Tandem OriT by Qu Mingzhi
mini-prep F-OriT_pSB1A2
- using Transgen mini plasmid puriflication kit.
- 30µL after purflication
mini-prep F-OriT_pSB1A2
- using Transgen mini plasmid puriflication kit.
- 30µL after purflication
mini-prep double digesting test result
- Digesting F-OriT_pSB1A2 with EcoR1/PstI.
- Digestion system contains:
1 µl 10*H 0.25 µl EcoRI 0.25 µl PstI 5 µl Plasmid 3.5 µl dH20 -------------------------- 10 µl Total
- 37℃ culutre for 3 hours.
- from left to right:
- mini-prep F-OriT_pSB1A2 -1 @ EcoRI/PstI
- mini-prep F-OriT_pSB1A2 -2 @ EcoRI/PstI
- Ori-T fragment
- pSB1A2 vector
- marker (DL2000 Plus)
Double digesting test for F-OriT_pSB1A2 & J23066
- use Double digesting test for gel extraction.
- F-OriT_pSB1A2 digestion system contains:
4 µl 10*H buffer 1 µl SpeI 1 µl PstI 20 µl Plasmid 14 µl dH20 -------------------------- 40 µl Total
- J23066 digestion system contains:
4 µl 10*M buffer 1 µl XbaI 1 µl PstI 20 µl Plasmid 14 µl dH20 -------------------------- 40 µl Total
electrophoresis result before gel extraction
- from left to right:
- F-OriT_pSB1A2 -1 @ SpeI/PstI
- F-OriT_pSB1A2 -2 @ SpeI/PstI
- J23066 -1 @ XbaI/PstI
- J23066 -2 @ XbaI/PstI
- marker (DL2000 plus)
electrophoresis result after gel extraction
- from left to right:
- F-OriT_pSB1A2 vector @ SpeI/PstI
- J23066 -2 fragment @ XbaI/PstI
- marker (DL2000 plus)
Ligation: F-OriT_pSB1A2 <- J23066(fragment)
Ligate the J23066 fragment and F-OriT_pSB1A2 vector
- Ligation system contains:
1 µl F-OriT_pSB1A2 vector 7 µl J23066 fragment 1 µl T4-Ligase 1 µl 10X T4 ligase buffer 0 µl dH20 -------------------------- 10 µl Total
- 16℃ overnight
Lock & Key By Yu Tao
oriT Knock Out
- By Xu Anting
oriT-deleted fragment purification
- Plasmid purification from overnight-incubated pUC18 strains.
- Digestion with restriction enzymes overnight.
- For strain F, keep the system in 30℃:
5 µl 10*K buffer 2 µl XbaI 25 µl Plasmid 18 µl ddH20 -------------------------- 50 µl Total
- For strain R751 and pSC101, keep them in 37℃:
7.5 µl 10*T buffer 1.5 µl XbaI 1.5 µl PstI 25 µl Plasmid 14.5 µl ddH20 -------------------------- 50 µl Total