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℃.