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 (I)

• 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

• 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. Maker(DL2000 plus)

• seems maker 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