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

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

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

• 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.