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

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℃.