IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-22

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=Tandem oriT by Qu Mingzhi=
==Amplification Culture of PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 (III)==
==Amplification Culture of PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 (III)==
*Amplification Culture of PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 from plates.
*Amplification Culture of PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 from plates.
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*PlacI-E0240 plasmid
*PlacI-E0240 plasmid
[[Image:Peking_2007-8-22_R0010gel_PlacI-I741051gel_E0240gel_PlacI-I741051plasmid_R0010plasmid.jpg‎ ]]
[[Image:Peking_2007-8-22_R0010gel_PlacI-I741051gel_E0240gel_PlacI-I741051plasmid_R0010plasmid.jpg‎ ]]
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 +
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=Lock & Key By Yu Tao=
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==J01008 Digestion Product Purification==
 +
*use Transgen EasyPure PCR Purification Kit.
 +
*50uL per tube after purflication, one tube, respectively.
 +
===Electrophorsis Result===
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*from ''left'' to ''right'':
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1-3:  51℃ purified pfu PCR product, digested pfu PCR product, digested taq PCR product
 +
4-7:  54℃ purified pfu PCR product, digested pfu PCR product, purified taq PCR product, digested taq PCR product
 +
8-11: 57℃ purified pfu PCR product, digested pfu PCR product, purified taq PCR product, digested taq PCR product
 +
12-15:60℃ purified pfu PCR product, digested pfu PCR product, purified taq PCR product, digested taq PCR product
 +
16:marker (DL2000 plus)
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17:  51℃ purified taq PCR product
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[[Image:Example.jpg]]
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*Comments: The bands are all smaller than 100bp. Not much hope.
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==Ligation: all the above digested PCR fragment==
 +
*Ligate all the fragments with R0010 vector, respectively.
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*Ligation system contains:
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<pre>
 +
7.5 µl    J01008 fragment
 +
1 µl      R0010 vector
 +
0.5 µl    Super T4-Ligase
 +
1 µl      10 X ligation buffer
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--------------------------
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10 µl      Total
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</pre>
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*The negative control group contains no fragment but ddH2O instead.
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*10min at 16℃。
 +
==Transformation: ==
 +
*Transform all ligation products into 100 µl DH5α competent cells.
 +
*Culture all cells at Amp+ LB plate for 12 hours.
 +
*Result to be seen tomorrow.

Revision as of 05:05, 31 August 2007

Contents

Tandem oriT by Qu Mingzhi

Amplification Culture of PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 (III)

  • Amplification Culture of PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 from plates.

mini-prep PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 (III)

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

mini-prep double digesting test

  • Digesting PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 with EcoRI/PstI.
  • Digestion system contains:
1    µl      10*H
0.25 µl      EcoRI
0.25 µl      PstI
5    µl      Plasmid
3.5  µl      dH20
--------------------------
40   µl      Total

electrophoresis result

from left to right:

  • PlacI-E0240 gel
  • PlacI-I741051-E0240 gel
  • E0240 gel
  • Marker(Marker I)
  • Marker(DL2000 plus)
  • PlacI-I741051-E0240 plasmid
  • PlacI-E0240 plasmid

Image:Peking_2007-8-22_R0010gel_PlacI-I741051gel_E0240gel_PlacI-I741051plasmid_R0010plasmid.jpg‎


Lock & Key By Yu Tao

J01008 Digestion Product Purification

  • use Transgen EasyPure PCR Purification Kit.
  • 50uL per tube after purflication, one tube, respectively.

Electrophorsis Result

  • from left to right:

1-3: 51℃ purified pfu PCR product, digested pfu PCR product, digested taq PCR product 4-7: 54℃ purified pfu PCR product, digested pfu PCR product, purified taq PCR product, digested taq PCR product 8-11: 57℃ purified pfu PCR product, digested pfu PCR product, purified taq PCR product, digested taq PCR product 12-15:60℃ purified pfu PCR product, digested pfu PCR product, purified taq PCR product, digested taq PCR product 16:marker (DL2000 plus) 17: 51℃ purified taq PCR product Image:Example.jpg

  • Comments: The bands are all smaller than 100bp. Not much hope.

Ligation: all the above digested PCR fragment

  • Ligate all the fragments with R0010 vector, respectively.
  • Ligation system contains:
7.5 µl     J01008 fragment
1 µl       R0010 vector
0.5 µl     Super T4-Ligase
1 µl       10 X ligation buffer
--------------------------
10 µl      Total
  • The negative control group contains no fragment but ddH2O instead.
  • 10min at 16℃。

Transformation:

  • Transform all ligation products into 100 µl DH5α competent cells.
  • Culture all cells at Amp+ LB plate for 12 hours.
  • Result to be seen tomorrow.
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