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

Tandem OriT by Qu Mingzhi, Ren Ze

mini-prep R-OriT, S-Orit (T-vector)

• 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)

Ligation: S-OriT -> pSB1A2, R-OriT -> pSB1A2

• Ligate the S-OriT fragment and pSB1A2 vector .
• Ligate the S-OriT fragment and pSB1A2 vector .
• use super fast T4 DNA ligase

super fast T4 DNA ligase Ligation system contains:

1   µl     vector
7   µl     E0240 fragment 
0.5 µl     ''super fast T4 DNA ligase''
1   µl     10X T4 ligase buffer
0.5 µl     dH20
--------------------------
10 µl     Total

transformation S-OriT -> pSB1A2, R-OriT -> pSB1A2

NEXT DAY: received 50+ clones, self-ligation did not grow.

Amplification Culture of PlacI-I741051 <- E0240, R0010 <- E0240

select Positive PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 Colonies from Plate, Culture in liquid LB, waiting for mini-prep.