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(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:

electrophoresis result after gel extraction

• from left to right:

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!.