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

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Contents

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)

Image:Peking_2007-8-2_OriT-pEasyT3_mimi-prep_result.jpg‎

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)

Image:Peking_2007-8-2_OriT-pEasy-T3_mini-prep_digesting_result.jpg‎

  • 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)

Image:Peking_2007-8-2_F-OriT_digesting_purification_result.jpg

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.
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