IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-17

Tandem OriT by Qu Mingzhi

mini-prep R-OriT, S-Orit

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

Double digesting test for R-OriT, S-OriT, E0040

• use Double digesting test for gel extraction.

• R-OriT digestion system contains(Standard Assembly fragment):

4 µl       10*H buffer
1 µl       EcoRI
1 µl       PstI
20 µl      Plasmid
14 µl      dH20
--------------------------
40 µl      Total

• S-OriT digestion system contains(Standard Assembly fragment):

4 µl       10*H buffer
1 µl       EcoRI
1 µl       PstI
20 µl      Plasmid
14 µl      dH20
--------------------------
40 µl      Total

• E0040 digestion system contains(Standard Assembly vector):

4 µl       10*H buffer
1 µl       EcoRI
1 µl       PstI
20 µl      Plasmid
14 µl      dH20
--------------------------
40 µl      Total

electrophoresis result before gel extraction

• from left to right:
  • 1-2 S-OriT @ EcoRI/PstI
  • 3-4 R-OriT @ EcoRI/PstI
  • 5-8 pSB1A2 @ EcoRI/PstI
  • 9 Maker (DL2000 plus)

electrophoresis result after gel extraction

• from left to right:

1. S-OriT
2. R-OriT
3. pSB1A2
4. pSB1A2
5. Maker (DL2000 plus)