IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-29

Lock & Key By Yu Tao and Zheng Qinsi

Double Digestion: B0015

• Digesting B0015 with XbaI/PstI.
• Each digestion system contains:

4 µl       10*M buffer
1 µl       XbaI
1 µl       PstI
20 µl      Plasmid
14 µl      ddH20
--------------------------
40 µl      Total

• 37℃ culutre for 6 hours.

Mini-prep: R0010, B0015, J23078(T vector)(2 colonies selected), R0010<-J23066(negative control group)

• Using Transgen mini plasmid purification kit.
• 50 uL per tube after purification, 2 tubes per type of plasmids.

Mini-prep Double Digesting Test Result

• Digesting R0010, B0015 and R0010<-J23066(negative control group) with EcoRI/PstI (H buffer) and J23078(T vector) PstI/XbaI (M buffer).
• Each digestion system contains：

1 µl       10*H/M buffer
0.25 µl    EcoRI/XbaI
0.25 µl    PstI
5 µl       Plasmid
3.5 µl     ddH20
--------------------------
10 µl      Total

• 37℃ culutre for 3 hours.
• from left to right:

1. R0010-4 plasmid
2. R0010-4 @ EcoRI/PstI
3. R0010-5 plasmid
4. R0010-5 @ EcoRI/PstI
5. B0015-3 plasmid
6. B0015-3 @ EcoRI/PstI
7. B0015-4 plasmid
8. B0015-4 @ EcoRI/PstI
9. J23078(T vector)-1-1 plasmid
10. J23078(T vector)-1-1 @ XbaI/PstI
11. J23078(T vector)-1-2 plasmid
12. J23078(T vector)-1-2 @ XbaI/PstI
13. marker (DL2000 Plus)

• from left to right:

1. J23078(T vector)-2-1 plasmid
2. J23078(T vector)-2-1 @ XbaI/PstI
3. J23078(T vector)-2-2 plasmid
4. J23078(T vector)-2-2 @ XbaI/PstI
5. R0010<-J23066(negative control group) plasmid
6. R0010<-J23066(negative control group) @ EcoRI/PstI
7. marker (DL2000 Plus)

• Comments: The purified plasmids are still mixed with unknown DNA(those bands between 1000bp and 2000bp), and we think it may be supercoiled plasmids.

oriT Knock Out

• By Xu Anting

Transformation results

1. All of the three products (F/R751/pSC101) have a high blue/white ratio. Maybe it is because I holded the ligating reaction for over 20 hours.
2. Forgot to do negative control.
3. dephosphorylation control has only 5 colony in the plate, in which there was only 1 blue spot. However, it was still hard to say whether the low colony number was because of the product loss in PCR kit purification, or due to the remained activity of CIAP (to deactivate CIAP mostly, 5 mM EDTA is required).

Sequencing of Products

• I sent a total number of 17 colonies for sequencing (Boshang Company), including 6 F, 7 R751 and 6 pSC101. I will get the result in a few days.