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