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

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
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
 * from left to right:
 * Comments: The bands are all smaller than 100bp. Not much hope.

Ligation: all the above digested PCR fragment
7.5 µl    J01008 fragment 1 µl      R0010 vector 0.5 µl    Super T4-Ligase 1 µl      10 X ligation buffer -- 10 µl     Total
 * Ligate all the fragments with R0010 vector, respectively.
 * Ligation system contains:
 * 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.