IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-27

=OriT Knock Out=
 * By Xu Anting

Enzyme Digestion for Vectors

 * I chose plasmid pUC18 as cloning vector. For plasmid digestion, only 5 hours of reaction is enough.


 * R751/pSC101 vector 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     Plasmid pUC18 20.5 µl   ddH20 -- 50 µl     Total


 * F vector digestion system contains:

5 µl      10*K buffer 1 µl      BamHI 20 µl     Plasmid pUC18 24 µl     ddH20 -- 50 µl     Total

Dephosphorylation of pUC18

 * Because F fragment requires single enzyme digestion, F vector has the same sticky ends in its two sides and hence has a high chance of self-ligation.
 * For pUC18 the "blue-white" screening could be used to distinguish self-ligated clones from products. However, to test the efficiency of my dephosphorylation protocol I set up a dephosphorylated control.
 * 1) Add 0.5 µL CIAP (calf intestinal alkaline phosphatase) in 10 uL digestion product.
 * 2) Bath in 37 centigrade for 30 min.
 * 3) Deactivation in 70 centigrade for 10 min.
 * 4) PCR kit purification to a final volumne of 10 uL.

Ligation of PCR fragments and pUC18

 * Ligation conditions: bathing in 4 centigrade for 12-16 hours.


 * Ligation system contains:

1 µl      10*T4 ligation buffer (Takara) 1 µl      T4 ligase (Takara) 4 µl      digested PCR fragment 4 µl      digested vector -- 10 µl     Total

=Lock & Key By Yu Tao and Zheng Qinsi=

Transformation result of R0010<-J23066

 * There are colonies both in the experimental plate and the negative control plate.
 * Colonies in the experimental plate are bigger, but both plates are of no differences in the number of colonies.
 * Select probable positive colonies from the experimental plate, culture them in liquid LB overday for mini-prep.

Mini-prep: R0010 and J23066

 * Using Transgen mini plasmid puriflication kit.
 * 50 uL after purflication.

mini-prep single digesting test result

 * Digesting R0010 and J23066 with EcoRI.
 * Each digestion system contains：

1 µl      10*H 0.5 µl    EcoRI 5 µl      Plasmid 3.5 µl    ddH20 -- 10 µl     Total


 * 37℃ culutre for 4 hours.
 * from left to right:
 * 1) R0010 plasmid
 * 2) R0010 @ EcoRI
 * 3) J23066 plasmid
 * 4) J23066 @ EcoRI
 * 5) marker (DL2000 Plus)
 * It seems that there are some impurities in the plasmids. I suggest we remini-prep all the plasmids.

Double Digestion: R0010, J23066 and R0040

 * Digesting R0010 and R0040 with SpeI/PstI, J23066 with PstI/XbaI.
 * Each digestion system contains:

2 µl      10*H 0.5 µl    SpeI/XbaI 0.5 µl    PstI 10 µl     Plasmid 7 µl      ddH20 -- 20 µl     Total


 * Treat R0010 with only one system but the other 2 plasmids with two systems.
 * 37℃ culutre for a whole day.
 * Results to be seen tomorrow.

Double Digestion: B0015 and E0040(pSB3K3)

 * Digesting B0015 with XbaI/PstI(M buffer) and SpeI/PstI(H buffer).
 * Each digestion system contains:

2 µl      10*H/M buffer 0.5 µl    XbaI/SpeI 0.5 µl    PstI 10 µl     Plasmid 7 µl      ddH20 -- 20 µl     Total


 * 37℃ culutre for overnight.
 * Treat each plasmid with 3 systems.

Mini-prep preparation: B0015, R0010 and J23066

 * As suggested, we do the mini-prep again.
 * Culture R0010 and J23066 positive colonies in liquid LB overnight for mini-prep.

Transformation: R0040<-J23078

 * Transform 10 µl R0040<-J23078 PCR product into 100 µl DH5α competent cells.
 * Culture all the cells at Amp+ LB plate for 12 hours.
 * Result to be seen tomorrow.

Ligation and transformation: J23078

 * Ligate the J23078 PCR product with the commercial pEASY-T2 T-vector (10mins done).
 * Transform 10 µl ligation product into 100 µl DH5α competent cells.
 * Culture all the cells at Amp+ LB plate for 12 hours.
 * Result to be seen tomorrow.

Mini-prep: R0010<-J23066

 * Using Transgen mini plasmid purification kit.
 * 50uL after purification.

Mini-prep single and double digesting test result

 * Digesting R0010<-J23078 with XbaI/PstI or only EcoRI.
 * Each single digestion system contains：

1 µl      10*M buffer 0.5 µl    EcoRI 5 µl      Plasmid 3 µl      ddH20 -- 10 µl     Total
 * Each doulbe digestion system contains：

1 µl      10*M buffer 0.5 µl    XbaI 0.5 µl    PstI 5 µl      Plasmid 3 µl      ddH20 -- 10 µl     Total
 * 37℃ culutre for 3 hours.
 * 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) marker (DL2000 Plus)
 * Suggest Yu Tao use R0010 @ PstI/SpeI and J23066 @ PstI/XbaI as control so that more information will be available.
 * The remaining samples are stored in -20℃.