IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-4

=Tandem OriT by Qu Mingzhi=

mini-prep F-OriT_pSB1A2

 * using Transgen mini plasmid puriflication kit.
 * 30µL after purflication

mini-prep F-OriT_pSB1A2

 * using Transgen mini plasmid puriflication kit.
 * 30µL after purflication

mini-prep double digesting test result
1 µl      10*H 0.25 µl   EcoRI 0.25 µl   PstI 5 µl      Plasmid 3.5 µl     dH20 -- 10 µl     Total
 * Digesting F-OriT_pSB1A2 with EcoR1/PstI.
 * Digestion system contains：
 * 37℃ culutre for 3 hours.
 * from left to right:
 * 1) mini-prep F-OriT_pSB1A2 -1 @ EcoRI/PstI
 * 2) mini-prep F-OriT_pSB1A2 -2 @ EcoRI/PstI
 * 3) Ori-T fragment
 * 4) pSB1A2 vector
 * 5) marker (DL2000 Plus)

Double digesting test for F-OriT_pSB1A2 & J23066
4 µl      10*H buffer 1 µl      SpeI 1 µl      PstI 20 µl     Plasmid 14 µl     dH20 -- 40 µl     Total 4 µl      10*M buffer 1 µl      XbaI 1 µl      PstI 20 µl     Plasmid 14 µl     dH20 -- 40 µl     Total
 * use Double digesting test for gel extraction.
 * F-OriT_pSB1A2 digestion system contains:
 * J23066 digestion system contains:

electrophoresis result before gel extraction

 * from left to right:
 * 1) F-OriT_pSB1A2 -1 @ SpeI/PstI
 * 2) F-OriT_pSB1A2 -2 @ SpeI/PstI
 * 3) J23066 -1       @ XbaI/PstI
 * 4) J23066 -2       @ XbaI/PstI
 * 5) marker （DL2000 plus)

electrophoresis result after gel extraction

 * from left to right:
 * 1) F-OriT_pSB1A2 vector @ SpeI/PstI
 * 2) J23066 -2  fragment @ XbaI/PstI
 * 3) marker （DL2000 plus)

Ligation: F-OriT_pSB1A2 <- J23066(fragment)
Ligate the J23066 fragment and F-OriT_pSB1A2 vector 1 µl     F-OriT_pSB1A2 vector 7 µl     J23066 fragment 1 µl     T4-Ligase 1 µl     10X T4 ligase buffer 0 µl     dH20 -- 10 µl    Total
 * Ligation system contains:
 * 16℃ overnight

=Lock & Key By Yu Tao=

=oriT Knock Out=
 * By Xu Anting

oriT-deleted fragment purification

 * 1) Plasmid purification from overnight-incubated pUC18 strains.
 * 2) Digestion with restriction enzymes overnight.
 * For strain F, keep the system in 30℃:

5 µl      10*K buffer 2 µl      XbaI 25 µl     Plasmid 18 µl     ddH20 -- 50 µl     Total


 * For strain R751 and pSC101, keep them in 37℃:

7.5 µl    10*T buffer 1.5 µl    XbaI 1.5 µl    PstI 25 µl     Plasmid 14.5 µl   ddH20 -- 50 µl     Total