IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-2: Difference between revisions

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*OriT by Qu Mingzhi
=OriT by Qu Mingzhi=
==mini-prep F-OriT_pEasy-T3==
==mini-prep F-OriT_pEasy-T3==
*using Transgen mini plasmid puriflication kit.
*using Transgen mini plasmid puriflication kit.
===mini-prep result===
*50µL after purflication
===mini-prep electrophorsis result===
*from ''left'' to ''right'':
*from ''left'' to ''right'':
#mini-prep F-OriT_pEasy-T3-1
#mini-prep F-OriT_pEasy-T3-1
#mini-prep F-OriT_pEasy-T3-2
#mini-prep F-OriT_pEasy-T3-2
#marker
#marker (DL2000 Plus)
[[Image:Peking_2007-8-2_OriT-pEasyT3_mimi-prep_result.jpg‎]]
[[Image:Peking_2007-8-2_OriT-pEasyT3_mimi-prep_result.jpg‎]]


===mini-prep double digesting test result===
===mini-prep double digesting test result===
*Digesting F-OriT_pEasy-T3 with EcoR1/PstI.  
*Digesting F-OriT_pEasy-T3 with EcoR1/PstI.  
*Digestion system contains (totally 10uL): 1uL 10*H, 0.25uL EcoRI, 0.25uL PstI, 5uL plasmid, 3.5uL ddH2O.
*Digestion system contains：
 
<pre>
1 µl      10*H
0.25 µl    EcoRI
0.25 µl    PstI
5 µl      Plasmid
3.5 µl      dH20
--------------------------
10 µl      Total
</pre>
*37℃ culutre for 3 hours.
*37℃ culutre for 3 hours.
*from ''left'' to ''right'':
*from ''left'' to ''right'':
#mini-prep F-OriT_pEasy-T3-1 @ EcoRI/PstI
#mini-prep F-OriT_pEasy-T3-1 @ EcoRI/PstI
#mini-prep F-OriT_pEasy-T3-2 @ EcoRI/PstI
#mini-prep F-OriT_pEasy-T3-2 @ EcoRI/PstI
#marker 1
#marker (DL2000 Plus)
#marker 2
#marker (DL2000 Plus)
[[Image:Peking_2007-8-2_OriT-pEasy-T3_mini-prep_digesting_result.jpg‎]]
[[Image:Peking_2007-8-2_OriT-pEasy-T3_mini-prep_digesting_result.jpg‎]]
*I think the channel is bright enough for purification,so.... ^_^
*I think the channel is bright enough for purification,so.... ^_^
Line 23: Line 34:
==Ori-T purification==
==Ori-T purification==
*use Transgen kit.  
*use Transgen kit.  
*30uL after purflication
*30µL after purflication
===electrophorsis result===
*from ''left'' to ''right'':
#purified F-Ori-T
#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.

Latest revision as of 06:54, 24 August 2007

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.
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