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

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!.

Lock & Key By Yu Tao

Transformation Result: Efficiency Test of Competent Cells III

• Result as expected.

                            | Competent Cells II | Competent Cells III | Competent Cells III
------------------------------------------------------------------------------------------------
           R0010            |        1 uL        |        1 uL         |       0 uL
------------------------------------------------------------------------------------------------
           ddH2O            |        0 uL        |        0 uL         |       1 uL
------------------------------------------------------------------------------------------------
antibiotics resistency test |        Amp         |        Amp          |  Amp/Kan,respectively
------------------------------------------------------------------------------------------------
      Number of clones      |      hundreds      |      thousands      |  none for both Amp/Kan

Transformation Result: R0040.J01010->E0040.B0015 and R0010.J01008<-B0015

• Only colonies in R0010.J01008<-B0015 experimental plate grow.
• Select probable positive colonies from the experimental plate, culture them in liquid LB overnight for mini-prep.
• We need to retry the R0040.J01010->E0040.B0015.

R0040.J01010 Digestion Product Purification

• Use Transgen Quick Gel Extraction Kit.
• Unfortunately, Qu just extracted the wrong band. As below:
• from left to right:

1. DL2000 plus marker
2. Digestion Product before purified.

• Qu extracted the larger(higher) band, but I need the smaller(lower) band. Do not matter, we can do it again.

Double Digestion: R0040.J01010

• The third time now.
• Digesting R0040.J01010 with EcoRI/SpeI, system components see previous record.
• 37℃ overnight.

Ligation and transformation: pEASY-T3<-J01008

• Retry the ligation.
• Use the pEASY-T3 clone vector.
• Transform the ligation product into DH5a competent cells.
• Result to be seen tonight.

Transformation Result

• Some clones grow.
• Select probable positive colonies from the experimental plate, culture them in liquid LB overnight for mini-prep.