IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-28
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oriT Knock Out
- By Xu Anting
- The transformation of ligation products finished, with incubating the resulting DH5a in LB Amp X-gal, 37 centigrade. Hoping to get results tomorrow.
Lock & Key By Yu Tao and Zheng Qinsi
Mini-prep: R0010, J23066 and B0015
- Using Transgen mini plasmid purification kit.
- 50uL per tube after purification, 2 tubes per type of plasmids.
Mini-prep single digesting test result
- Digesting all the plasmids with EcoRI.
- Each digestion system contains:
1 µl 10*H buffer 0.25 µl EcoRI 3 µl Plasmid 6 µl ddH20 -------------------------- 10 µl Total
- 37℃ culutre for 3 hours.
- from left to right:
- $$$ @ EcoRI
- marker (DL2000 Plus)
Mini-prep double digesting test result
- Samples stored at -20℃ last night.
- from left to right:
- R0010<-J23066-1 @ EcoRI
- R0010<-J23066-1 @ XbaI/PstI
- R0010<-J23066-2 @ EcoRI
- R0010<-J23066-2 @ XbaI/PstI
- R0010<-J23066-3 @ EcoRI
- R0010<-J23066-3 @ XbaI/PstI
- R0010 @ PstI/SpeI
- J23066 @ PstI/XbaI
- marker (DL2000 Plus)
- Still there are some unknown bands shown up in R0010 and B0015 plasmids. I suggest a re-mini-prep again.
B0015, E0040(pSB3K3) and R0040 Double Digestion Product Purification
- The digestion was under way yesterday.
- use Transgen EasyPure PCR Purification Kit for E0040(pSB3K3) and Quick Gel Extraction Kit for B0015.
- 30uL per tube after purflication, one tube, respectively.
Electrophorsis Result
- from left to right:
- marker
Ligation: E0040(pSB3K3)<-B0015
- Ligate the B0015 fragment and E0040(pS3K3) vector
- Ligation system contains:
7 µl B0015 fragment 1 µl E0040(pSB3K3) vector 1 µl T4-Ligase 1 µl 10 X ligation buffer 0 µl ddH20 -------------------------- 10 µl Total
- The negative control group contains no fragment but 7uL ddH2O instead.
- 4℃ overnight.
Transformation Result: J23078(pEASY-T2)
- There are colonies both in the experimental plate and the negative control plate.
- Both plates are of no differences in the number of colonies.
- Number of colonies: 5 in the experimental plate and 2 in the negative control plate.
- Select 2 probable positive colonies from the experimental plate, culture them in liquid LB overnight for mini-prep.
Mini-prep Preparation: R0010 and B0015
- Culture R0010 and B0015 positive colonies in liquid LB overnight for mini-prep.