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
1   µl      10*H 0.25 µl     EcoRI 0.25 µl     PstI 5   µl      Plasmid 3.5 µl      dH20 -- 40  µl      Total
 * Digesting E0240 with EcoRI/PstI.
 * E0240 Digestion system contains：

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.

4 µl      10*H buffer 1 µl      SpeI 1 µl      PstI 20 µl     Plasmid 14 µl     dH20 -- 40 µl     Total
 * 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*M buffer 1 µl      XbaI 1 µl      PstI 20 µl     Plasmid 14 µl     dH20 -- 40 µl     Total
 * E0240 digestion system contains(Standard Assembly vector):

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
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
 * 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:

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
| Competent Cells II | Competent Cells III | Competent Cells III
 * Result as expected.

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.