IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-20

Tandem OriT by Qu Mingzhi & Ren Ze

Preparation of Competent Cells: F+, R751

• Preparation F+, R751 competent cell for Conjugation test.

Comptent Cells efficiency test

• Transformation test

Competent Cell/plasmid      transformation growth

F+/- (Nagetive control)             -
F+/Ori-T_pSB1A2                    50+
F+/R0010(Postive control)          100+
-----------------------------------------------
R751/-(Nagetive control)            -
R751/pSC101                        10+
R751/R0010(Postive control)        50+

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

• last mini-prep digesting test didn't show the correct backbone.
• see:IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-18
• Amplification Culture of PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 from plates.
• 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 Marker(DL2000 plus)
  • 2-3 PlacI-E0240 @ EcoRI/PstI
  • 4-5 PlacI-I741051-E0240 @ EcoRI/PstI

Lock & Key By Yu Tao

Mini-prep: R0010<-J01008(1-6) and R0040.J01010->E0040.B0015(1-3)

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

Mini-prep Double Digesting Test Result

• Digesting all plasmids above, R0010, E0040.B0015 and E0040 with EcoRI/PstI.
• Each digestion system contains：

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

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

1. R0010<-J01008-1 @ EcoRI/PstI
2. R0010<-J01008-2 @ EcoRI/PstI
3. R0010<-J01008-3 @ EcoRI/PstI
4. R0010<-J01008-4 @ EcoRI/PstI
5. R0010<-J01008-5 @ EcoRI/PstI
6. R0010<-J01008-6 @ EcoRI/PstI
7. R0010 @ EcoRI/PstI
8. R0040.J01010->E0040.B0015-1 @ EcoRI/PstI
9. R0040.J01010->E0040.B0015-2 @ EcoRI/PstI
10. R0040.J01010->E0040.B0015-3 @ EcoRI/PstI
11. E0040.B0015 @ EcoRI/PstI
12. E0040 @ EcoRI/PstI
13. marker (DL2000 Plus)

• Conclusion:

1. R0040.J01010.E0040.B0015 makes it.
2. R0010<-J01008 fails.

• Stripe the R0040.J01010.E0040.B0015-2 on Kan+ LB plate for storage.

Sequencing

• Send the following precultures to Invitrogen for sequencing.

7. E0040.B0015 pSB3K3 8. I74051 pSB1A2 (Qu) 9. R-oriT pSB1A2 (Qu) 10. S-oriT pSB1A2 (Qu) 11. R0040.J01010 pSB1A2 12. T-J01010 pEASY-T3 13. T-J01008 pEASY-T3

• Use vf2 and vr primer for pSB plasmids and T7 primer for T3 plasmids.

Ligation: R0010<-J01008-2-1,2

• Ligate the J01008-2-1 and 2 fragment and R0010 vector
• Ligation system contains:

3 µl       R0040.J01010 fragment
0.5 µl     E0040.B0015 vector
0.5 µl     Super T4-Ligase
1 µl       10 X ligation buffer
5 µl       ddH20
--------------------------
10 µl      Total

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

Transformation:

• Transform all ligation products into 100 µl DH5α competent cells.
• Culture all cells at Amp+ LB plate for 12 hours.
• Result to be seen tomorrow.