IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-16

Tandem OriT by Qu Mingzhi

Amplification Culture of Ori-T

• select Positive OriT-pEASY-3 Colonies from Plate,Culture in liquid LB,waiting for mini-prep.

mini-prep E0240

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

mini-prep double digesting test

• Digesting E0240 with EcoRI/PstI.
• E0240 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. E0240 -1 @ EcoRI/PstI
2. E0240 -2 @ EcoRI/PstI
3. pSB1A2
4. Maker(DL2000 plus)

Double digesting test for PlacI-I741051 & R0010 & E0240

• use Double digesting test for gel extraction.

• PlacI-I74051 digestion system contains(as Standard assembly vector):

4 µl       10*M buffer
1 µl       EcoRI
1 µl       XbaI
20 µl      Plasmid
14 µl      dH20
--------------------------
40 µl      Total