IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-28

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:
 * 1) $$$ @ EcoRI
 * 2) marker (DL2000 Plus)

Mini-prep double digesting test result

 * Samples stored at -20℃ last night.
 * from left to right:
 * 1) R0010<-J23066-1 @ EcoRI
 * 2) R0010<-J23066-1 @ XbaI/PstI
 * 3) R0010<-J23066-2 @ EcoRI
 * 4) R0010<-J23066-2 @ XbaI/PstI
 * 5) R0010<-J23066-3 @ EcoRI
 * 6) R0010<-J23066-3 @ XbaI/PstI
 * 7) R0010 @ PstI/SpeI
 * 8) J23066 @ PstI/XbaI
 * 9) 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:
 * 1) marker
 * 1) 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.