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.

Lock & Key By Yu Tao

Mini-prep: T-J01008 and R0010.J01008<-B0015

• Using Transgen mini plasmid purification kit.
• 50uL per tube after purification, 2 tubes per type of plasmids.

Mini-prep Double Digesting Test Result

• Digesting all plasmids above and R0010.J01008(as negative control) with EcoRI/PstI.
• Each digestion system contains：

1 µl       10*H buffer
0.25 µl    EcoRI
0.25 µl    PstI
3 µl       Plasmid
5.5 µl     ddH20
--------------------------
10 µl      Total

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

1. T-J01008-1 @ EcoRI/PstI
2. T-J01008-2 @ EcoRI/PstI
3. R0010.J01008<-B0015-1 @ EcoRI/PstI
4. R0010.J01008<-B0015-2 @ EcoRI/PstI
5. R0010.J01008 @ EcoRI/PstI
6. marker (DL2000 Plus)

• Conclusion: We can see the J01008 fragment. However, R0010.J01008 fragment band is smaller than 200bp band, which means the previously ligated R0010<-J01008 might be wrong.

R0040.J01010 Digestion Product Purification

• use Transgen Quick Gel Extraction Kit.
• 30uL per tube after purflication, one tube, respectively.

Electrophorsis Result

• from left to right:

1. R0040.J01010 @ EcoRI/SpeI purified digestion product
2. marker (DL2000 plus)

Ligation: R0040.J01010<-E0040.B0015

• Ligate the R0040.J01010 fragment and E0040.B0015 vector
• Ligation system contains:

7 µl       R0040.J01010 fragment
1 µl       E0040.B0015 vector
0.5 µl     Super T4-Ligase
1 µl       10 X ligation buffer
0.5 µl     ddH20
--------------------------
10 µl      Total

• The negative control group contains no fragment but ddH2O instead.
• 10min at 16℃

Transformation: R0040.J01010<-E0040.B0015 ligation product

• Transform all ligation product into 100 µl DH5α competent cells.
• Culture all cells at Amp+ LB plate for 12 hours.
• Result to be seen tomorrow.

Double Digestion: R0040.J01010 and E0040.B0015

• Digesting R0040.J01010 with EcoRI/SpeI and E0040.B0015 with EcoRI/XbaI.
• Each digestion system contains:

For R0040.J01010                For E0040.B0015
2 µl       10*H           /     2 µl       10*M
0.5 µl     EcoRI          /     0.5 µl     EcoRI
0.5 µl     SpeI           /     0.5 µl     XbaI
17 µl      Plasmid        /     10 µl      Plasmid
0 µl       ddH2O          /     7 µl       ddH2O 
----------------------------------------------------
20 µl      Total          /     20 µl      Total  

• 37℃ overnight.

R0040.J01010 and E0040.B0015 Digestion Product Purification

• Use Transgen EasyPure PCR Purification Kit for E0040.B0015 and Quick Gel Extraction Kit for R0040.J01010.
• 30uL per tube after purflication, one tube, respectively.

Electrophorsis Result

• from left to right:

1. E0040.B0015 @ EcoRI/XbaI purified digestion product
2. R0040.J01010 @ EcoRI/SpeI purified digestion product
3. R0040 @ EcoRI/PstI
4. R0040.J01010 @ EcoRI/PstI
5. marker (DL2000 plus)

Mini-prep Preparation: R0010, R0040.J01010 and E0040.B0015

• Culture positive colonies in liquid LB overnight for mini-prep.