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

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.