IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-26

=Lock & Key By Yu Tao=

Double Digesting J23078

 * Digesting J23078 PCR product with EcoR1/PstI.
 * Digestion system contains:

2 µl      10*M buffer 0.5 µl    XbaI 0.5 µl    PstI 10 µl     PCR product 7 µl      ddH20 -- 20 µl     Total


 * 37℃ culture for 9 hours and 16℃ culture for 2 hours.

J23078 and R0040 Digestion Product purification

 * use Transgen EasyPure PCR Purification Kit for J23078 and Quick Gel Extraction Kit for R0040.
 * 30uL after purflication, respectively.

electrophorsis result

 * from left to right:
 * 1) J23078 @ XbaI/PstI without Genefinder(DNA dye) but loading buffer newly prepared by ourselves added
 * 2) J23078 @ XbaI/PstI without Genefinder(DNA dye) but loading buffer newly prepared by ourselves added
 * 3) J23078 @ XbaI/PstI with Genefinder(DNA dye) and loading buffer newly prepared by ourselves added
 * 4) J23078 @ XbaI/PstI with Genefinder(DNA dye) and loading buffer newly prepared by ourselves added
 * 5) J23078 @ XbaI/PstI without Genefinder(DNA dye) but commercial loading buffer prepared by ourselves added
 * 6) J23078 @ XbaI/PstI without Genefinder(DNA dye) but commercial loading buffer prepared by ourselves added
 * 7) Precise Quantified MarkerI
 * 8) R0040 @ PstI/SpeI
 * Results are expected.
 * I am determined to use the commercial loading buffer and to add DNA dye to the samples everytime from now.

Ligation: J23078(crRNA) and R0040

 * Ligate the J23078 fragment and R0040 vector
 * Ligation system contains:

1 µl      J23078 fragment 7 µl      R0040 vector 1 µl      T4-Ligase 1 µl      10 X ligation buffer 0 µl      ddH20 -- 10 µl     Total


 * 4℃ overnight(11 hours), then to be at 16℃ for 6 hours.

Transformation: R0010<-J23066

 * Transform 10 µl R0010<-J23066 ligation product into 100 µl DH5αcompetent cells.
 * Culture all the cells at Amp+ LB plate for 12 hours.
 * Results to be seen tommorow.

Mini-prep preparation: R0010 and J23066

 * Culture R0010 and J23066 positive colonies in liquid LB overnight for mini-prep.

=oriT Knock Out=
 * By Xu Anting

Enzyme Digestion for PCR Fragments

 * 1) I chose BamHI and SalI as restriction sites for cloning fragments of R751 and pSC101. As for F, only BamHI site is available.  The choice is based on the multiple cloning sites in plasmid pKO3: only BamHI, SalI, SmaI and NotI are available.
 * 2) SalI digestion for PCR products requires 20 hours in 37 centigrade, but BamHI is not so stable in this degree. So the experimental condition is somewhat tricky: I add half of required BamHI first and another half 10 hours later.
 * R751/pSC101 digestion system contains:

7.5 µl    10*T buffer (to a final concentration of 1.5*T) 1 µl      BamHI 1 µl      SalI 20 µl     PCR product 20.5 µl   ddH20 -- 50 µl     Total


 * F digestion system contains (hold in 30 centigrade):

5 µl      5*K buffer 1 µl      BamHI 20 µl     PCR product 24 µl     ddH20 -- 50 µl     Total