IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-2

=OriT by Qu Mingzhi=

mini-prep F-OriT_pEasy-T3

 * using Transgen mini plasmid puriflication kit.
 * 50µL after purflication

mini-prep electrophorsis result

 * from left to right:
 * 1) mini-prep F-OriT_pEasy-T3-1
 * 2) mini-prep F-OriT_pEasy-T3-2
 * 3) marker (DL2000 Plus)

mini-prep double digesting test result

 * Digesting F-OriT_pEasy-T3 with EcoR1/PstI.
 * Digestion system contains：

1 µl      10*H 0.25 µl   EcoRI 0.25 µl   PstI 5 µl      Plasmid 3.5 µl     dH20 -- 10 µl     Total
 * 37℃ culutre for 3 hours.
 * from left to right:
 * 1) mini-prep F-OriT_pEasy-T3-1 @ EcoRI/PstI
 * 2) mini-prep F-OriT_pEasy-T3-2 @ EcoRI/PstI
 * 3) marker (DL2000 Plus)
 * 4) marker (DL2000 Plus)
 * I think the channel is bright enough for purification,so.... ^_^

Ori-T purification

 * use Transgen kit.
 * 30µL after purflication

electrophorsis result

 * from left to right:
 * 1) purified F-Ori-T
 * 2) marker (DL2000 Plus)

Ligation:F-OriT ->pSB1A2

 * Ligate the F-Ori-T fragment and pSB1A2 vector
 * Ligation system contains (totally 10uL):3uL Ori-T, 2uL pSB1A2, 1uL T4-Ligase, 1uL 10Xbuffer, 3uL dH20
 * 4℃ overnight

=Lock & Key By Yu Tao=

=oriT Knock Out=
 * By Xu Anting

Competent cell test

 * Transform pUC18 into three competent strains, and spread them onto Amp+ LB plates (with X-gal added).
 * Set negative control as spreading strains without pUC18 onto Amp+ LB plates.
 * Results: strain F has ~50 colonies, while R751 and pSC101 have 200+ colonies.