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

From OpenWetWare

Jump to: navigation, search

Contents

Tandem oriT by Mingzhi Qu

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.
Personal tools