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

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Contents

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

  • Digesting F-OriT_pSB1A2 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_pSB1A2 -1 @ EcoRI/PstI
  2. mini-prep F-OriT_pSB1A2 -2 @ EcoRI/PstI
  3. Ori-T fragment
  4. pSB1A2 vector
  5. marker (DL2000 Plus)

Image:Peking_2007-8-4_Ori-T_pSB1A2mini-prep_double_digesting_test_result(small).jpg‎

Double digesting test for F-OriT_pSB1A2 & J23066

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

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)

Image:Peking_2007-8-4_OriT-pSB1A2_J23066_digestion.jpg‎

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)

Image:Peking_2007-8-4_OriTpSB1A2_vector_J23066_fragment_after_gel_extraction.jpg‎

Ligation: F-OriT_pSB1A2 <- J23066(fragment)

Ligate the J23066 fragment and F-OriT_pSB1A2 vector

  • Ligation system contains:
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
  • 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
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