IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-18

Tandem Ori-T by Qu Mingzhi, Ren Ze

Amplification Culture of R-OriT-pSB1A2 & S-OriT-pSB1A2

• select Positive R-OriT & S-OriT Colonies from Plate,Culture in liquid LB, waiting for mini-prep.

mini-prep PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 (I)

• using Transgen mini plasmid puriflication kit.
• 50µL after purflication.

mini-prep double digesting test

• Digesting PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 with EcoRI/PstI.
• Digestion system contains：

1    µl      10*H
0.25 µl      EcoRI
0.25 µl      PstI
5    µl      Plasmid
3.5  µl      dH20
--------------------------
40   µl      Total

electrophoresis result

• from left to right:

1. PlacI-E0240 @ EcoRI/PstI
2. PlacI-E0240 @ EcoRI/PstI
3. PlacI-I741051_E0240 @ EcoRI/PstI
4. PlacI-I741051_E0240 @ EcoRI/PstI
5. Marker(DL2000 plus)

• seems marker have some problem ,do a digesting test again.

electrophoresis result 2

• PlacI-I741051_E0240 did not show the correct backbone.

electrophoresis result 3

• from left to right:

1. pSB1A2 fragment
2. R0010 fragment
3. E0240 @ EcoRI/PstI
4. Maker(DL2000 plus)
5. R0010-E0240 @ EcoRI/PstI
6. R0010-I74051-E0240 @ EcoRI/PstI
7. R0010-I74051 @ EcoRI/PstI

• still have the problem! need to do it again.

Lock & Key By Yu Tao

Transformation Result: R0040.J01010->E0040.B0015

• There are colonies both in the experimental plate and the negative control plate.
• Number of colonies: both plates exist tens of clones, and the experimental plate contains more.
• Select probable positive colonies from the experimental and the negative control plate, culture them in liquid LB overnight for mini-prep.

Mini-prep: R0010, R0040.J01010 and E0040.B0015

• The precultured R0010 DH5a seems contaminated, so put it aside.
• Using Transgen mini plasmid purification kit.
• 50uL per tube after purification, 3 tubes per type of plasmids.

Mini-prep Double Digesting Test Result

• Digesting all plasmids above with EcoRI/PstI.
• Each digestion system contains：

1 µl       10*H buffer
0.25 µl    EcoRI
0.25 µl    PstI
3 µl       Plasmid
3.5 µl     ddH20
--------------------------
10 µl      Total

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

1. R0010 @ EcoRI/PstI
2. R0010.J01008 @ EcoRI/PstI
3. R0040 @ EcoRI/PstI
4. R0040.J01010 @ EcoRI/PstI
5. marker (DL2000 Plus)

• from left to right:

1. E0040 @ EcoRI/PstI
2. E0040.B0015-1 @ EcoRI/PstI
3. E0040.B0015-2 @ EcoRI/PstI
4. E0040.B0015-3 @ EcoRI/PstI
5. R0040 @ EcoRI/PstI
6. R0040.J01010-1 @ EcoRI/PstI
7. R0040.J01010-2 @ EcoRI/PstI
8. R0040.J01010-3 @ EcoRI/PstI
9. marker (DL2000 Plus)

• Conclusion:

1. R0040.J01010 fragment is larger than R0040, which means the J01010 is really ligated to R0040. However, R0010.J01008 fragment is as large as R0010, so the failure of this ligation is confirmed.
2. In the second electrophoresis result, the E0040.B0015 fragments seems a little bit larger than E0040, but it still requires a reconfirmation. The newly minipreped R0040.J01010 is correct.

Ligation: R0010<-J01008 and R0040.J01010->E0040.B0015

• Ligate the J01008 fragment and R0010 vector, as well as R0040.J01010 fragment and E0040.B0015 vector.
• Ligation system contains:

7.5 µl     R0040.J01010 fragment   /   8 µl       R0040.J01010 fragment
1 µl       E0040.B0015 vector      /   0.5 µl     E0040.B0015 vector  
0.5 µl     Super T4-Ligase
1 µl       10 X ligation buffer
--------------------------
10 µl      Total

• The negative control group contains no fragment but ddH2O instead.
• 10min at 16℃.

Transformation:

• Transform all ligation product into 100 µl DH5α competent cells.
• Culture all R0010<-J01008 cells at Amp+ LB plate and all R0040.J01010->E0040.B0015 cells at Kan+ LB plate for 12 hours.
• Result to be seen tomorrow.

Mini-prep Preparation: R0010 and R0010<-J01008

• Because the R0010 preculture yesterday seems contaminated, I reculture R0010 positive colonies in liquid LB overnight for mini-prep.
• Also select 6 clones from the previous R0010<-J01008 plate for mini-prep to find whether there are any positive clones.