IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-15

=Tandem OriT by Qu Mingzhi=

transformation R-OriT & S-OriT

 * Use pEASY-3 as cloning vector .(Amp+)
 * tramsformation system contains :3uL R-OriT, S-OriT PCR product(after purflication), 1uL pEasy-T3
 * the Culture Dish(LB/Amp+) are trated with 5uL 1M IPTG & 40uL 40mg/mL x-Gal.
 * NEXT DAY: 10+ white cloney received.

Amplification Culture of E0240

 * select Positive E0240 Colonies from Plate,Culture in liquid LB,waiting for mini-prep.

=Lock & Key By Yu Tao=

R0010.J01008 and R0040.J01010 Digestion Product Purification

 * Use Transgen EasyPure PCR Purification Kit for R0010<-J01008 and Quick Gel Extraction Kit for R0040<--J01010.
 * 30uL per tube after purflication, one tube, respectively.

Electrophorsis Result

 * from left to right:
 * 1) DL 2000 plus marker
 * 2) R0010.J01008 @ SpeI/PstI purified digestion product
 * 3) R0040.J01010 @ EcoRI/SpeI purified digestion product

Recheck previous B0015 fragment and E0040.B0015 vector result

 * from left to right:
 * 1) B0015-1 @ XbaI/PstI purified digestion product
 * 2) B0015-2 @ XbaI/PstI purified digestion product
 * 3) E0040.B0015 @ EcoR/XbaI purified digestion product
 * 4) DL2000 plus marker
 * Conclusion: Use the B0015-2 @ XbaI/PstI purified digestion product and E0040.B0015 @ EcoR/XbaI purified digestion product.


 * Ligate the R0040.J01010 fragment and E0040.B0015 vector, as well as the B0015 fragment and R0010<-J01008 vector.
 * Ligation system contains:

7 µl      R0040.J01010 fragment   /   3 µl       B0015 fragment 1 µl      E0040.B0015 vector      /   1 µl       R0010<-J01008 vector 0.5 µl    Super T4-Ligase 1 µl      10 X ligation buffer 0 µl      ddH20                   /   4 µl       ddH20 -- 9.5 µl    Total


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

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

 * Transform all ligation products into 100 µl DH5α competent cells.
 * Culture all R0010.J01008<-B0015 cells at Amp+ LB plate and all R0040.J01010->E0040.B0015 cells at Kan+ LB plate for 12 hours.
 * Result to be seen tomorrow.

New competent cells preparation

 * Prepare Competent Cells III

Competent Cells III efficiency test
| Competent Cells II | Competent Cells III | Competent Cells III
 * Trasform 1uL R0010 plasmid into competent cells for test.

R0010           |        1 uL        |        1 uL         |       0 uL

ddH2O           |        0 uL        |        0 uL         |       1 uL

antibiotics resistency test |       Amp         |        Amp          |  Amp/Kan,respectively

Double Digestion: R0040.J01010

 * Because the purified digestion product of R0040.J01010 is almost unseen in the electrophoresis result, I decide to redo it once more.
 * Digesting R0040.J01010 with EcoRI/SpeI.
 * Each digestion system contains:

4 µl      10*H 1 µl      EcoRI 1 µl      SpeI 25 µl     Plasmid 9 µl      ddH20 -- 40 µl     Total


 * 37℃ culutre overnight.