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

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Tandem OriT by Qu Mingzhi

Amplification Culture of R-OriT & S-OriT(T-vector)

  • select Positive R-OriT & S-OriT 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(Standard Assembly vector):
4 µl       10*H buffer
1 µl       SpeI
1 µl       PstI
20 µl      Plasmid
14 µl      dH20
--------------------------
40 µl      Total
  • R0010 digestion system contains(Standard Assembly vector):
4 µl       10*H buffer
1 µl       SpeI
1 µl       PstI
20 µl      Plasmid
14 µl      dH20
--------------------------
40 µl      Total
  • E0240 digestion system contains(Standard Assembly vector):
4 µl       10*M buffer
1 µl       XbaI
1 µl       PstI
20 µl      Plasmid
14 µl      dH20
--------------------------
40 µl      Total

electrophoresis result before gel extraction

  • from left to right:
    • 1-2 PlacI-I741051 @ SpeI/PstI
    • 3-4 R0010 @ SpeI/PstI
    • 5-8 E0240 @ PstI/XbaI
    • 9 Marker(DL2000 plus)

electrophoresis result after gel extraction

  • from left to right:
  1. PlacI-I741051
  2. R0010
  3. pSB1A2
  4. pSB1A2

Ligation: PlacI-I741051 <- E0240 & R0010 <- E0240

  • Ligate the E0240 fragment and PlacI-I741051 vector .
  • Ligate the E0240 fragment and R0010 vector .
  • use super fast T4 DNA ligase
  • super fast T4 DNA ligase Ligation system contains:
2   µl     vector
3   µl     E0240 fragment 
0.5 µl     ''super fast T4 DNA ligase''
1   µl     10X T4 ligase buffer
3.5 µl     dH20
--------------------------
10 µl     Total

transformation PlacI-I741051 <- E0240 & R0010 <- E0240

  • NEXT DAY: Received clones...R0010 <- E0240 shows green GFP!.