IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-4

Tandem OriT by Qu Mingzhi

mini-prep F-OriT_pSB1A2

• using Transgen mini plasmid puriflication kit.
• 30µL after purflication

mini-prep F-OriT_pSB1A2

• using Transgen mini plasmid puriflication kit.
• 30µL after purflication

mini-prep double digesting test result

• Digesting F-OriT_pSB1A2 with EcoR1/PstI.
• Digestion system contains：

1 µl       10*H
0.25 µl    EcoRI
0.25 µl    PstI
5 µl       Plasmid
3.5 µl      dH20
--------------------------
10 µl      Total

• 37℃ culutre for 3 hours.
• from left to right:

1. mini-prep F-OriT_pSB1A2 -1 @ EcoRI/PstI
2. mini-prep F-OriT_pSB1A2 -2 @ EcoRI/PstI
3. Ori-T fragment
4. pSB1A2 vector
5. marker (DL2000 Plus)

Double digesting test for F-OriT_pSB1A2 & J23066

• use Double digesting test for gel extraction.
• F-OriT_pSB1A2 digestion system contains:

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

• J23066 digestion system contains:

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. F-OriT_pSB1A2 -1 @ SpeI/PstI
2. F-OriT_pSB1A2 -2 @ SpeI/PstI
3. J23066 -1 @ XbaI/PstI
4. J23066 -2 @ XbaI/PstI
5. marker （DL2000 plus)

electrophoresis result after gel extraction

• from left to right:

1. F-OriT_pSB1A2 vector @ SpeI/PstI
2. J23066 -2 fragment @ XbaI/PstI
3. marker （DL2000 plus)

Ligation: F-OriT_pSB1A2 <- J23066(fragment)

Ligate the J23066 fragment and F-OriT_pSB1A2 vector

• Ligation system contains:

1  µl     F-OriT_pSB1A2 vector
7  µl     J23066 fragment 
1  µl     T4-Ligase
1  µl     10X T4 ligase buffer
0  µl     dH20
--------------------------
10 µl     Total

• 16℃ overnight

oriT Knock Out

• By Xu Anting

oriT-deleted fragment purification

1. Plasmid purification from overnight-incubated pUC18 strains.
2. Digestion with restriction enzymes overnight.

For strain F, keep the system in 30℃:

5 µl       10*K buffer
2 µl       XbaI
25 µl      Plasmid
18 µl      ddH20
--------------------------
50 µl      Total

For strain R751 and pSC101, keep them in 37℃:

7.5 µl     10*T buffer
1.5 µl     XbaI
1.5 µl     PstI
25 µl      Plasmid
14.5 µl    ddH20
--------------------------
50 µl      Total