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.

4 µl      10*H buffer 1 µl      EcoRI 1 µl      PstI 20 µl     Plasmid 14 µl     dH20 -- 40 µl     Total
 * 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):

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
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
 * Ligate the S-OriT fragment and pSB1A2 vector.
 * Ligate the S-OriT fragment and pSB1A2 vector.
 * use super fast T4 DNA ligase

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.