IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-20

=Tandem OriT by Qu Mingzhi & Ren Ze=

Preparation of Competent Cells: F+, R751

 * Preparation F+, R751 competent cell for Conjugation test.

Comptent Cells efficiency test
Competent Cell/plasmid     transformation growth
 * Transformation test

F+/- (Nagetive control)            - F+/Ori-T_pSB1A2                   50+ F+/R0010(Postive control)         100+ --- R751/-(Nagetive control)           - R751/pSC101                       10+ R751/R0010(Postive control)       50+

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

 * last mini-prep digesting test didn't show the correct backbone.
 * see:IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-18
 * Amplification Culture of PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 from plates.
 * 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:
 * 1 Marker(DL2000 plus)
 * 2-3 PlacI-E0240 @ EcoRI/PstI
 * 4-5 PlacI-I741051-E0240 @ EcoRI/PstI

=Lock & Key By Yu Tao=

Mini-prep: R0010<-J01008(1-6) and R0040.J01010->E0040.B0015(1-3)

 * Using Transgen mini plasmid purification kit.
 * 50uL per tube after purification, 2 tubes per type of plasmids.

Mini-prep Double Digesting Test Result
1 µl      10*H buffer 0.25 µl   EcoRI 0.25 µl   PstI 3 µl      Plasmid 5.5 µl    ddH20 -- 10 µl     Total
 * Digesting all plasmids above, R0010, E0040.B0015 and E0040 with EcoRI/PstI.
 * Each digestion system contains：
 * 37℃ culutre for 3 hours.
 * from left to right:
 * 1) R0010<-J01008-1 @ EcoRI/PstI
 * 2) R0010<-J01008-2 @ EcoRI/PstI
 * 3) R0010<-J01008-3 @ EcoRI/PstI
 * 4) R0010<-J01008-4 @ EcoRI/PstI
 * 5) R0010<-J01008-5 @ EcoRI/PstI
 * 6) R0010<-J01008-6 @ EcoRI/PstI
 * 7) R0010 @ EcoRI/PstI
 * 8) R0040.J01010->E0040.B0015-1 @ EcoRI/PstI
 * 9) R0040.J01010->E0040.B0015-2 @ EcoRI/PstI
 * 10) R0040.J01010->E0040.B0015-3 @ EcoRI/PstI
 * 11) E0040.B0015 @ EcoRI/PstI
 * 12) E0040 @ EcoRI/PstI
 * 13) marker (DL2000 Plus)
 * Conclusion:
 * 1) R0040.J01010.E0040.B0015 makes it.
 * 2) R0010<-J01008 fails.
 * Stripe the R0040.J01010.E0040.B0015-2 on Kan+ LB plate for storage.

Sequencing
7. E0040.B0015 pSB3K3 8. I74051 pSB1A2 (Qu) 9. R-oriT pSB1A2 (Qu) 10. S-oriT pSB1A2 (Qu) 11. R0040.J01010 pSB1A2 12. T-J01010 pEASY-T3 13. T-J01008 pEASY-T3
 * Send the following precultures to Invitrogen for sequencing.
 * Use vf2 and vr primer for pSB plasmids and T7 primer for T3 plasmids.

Ligation: R0010<-J01008-2-1,2
3 µl      R0040.J01010 fragment 0.5 µl    E0040.B0015 vector 0.5 µl    Super T4-Ligase 1 µl      10 X ligation buffer 5 µl      ddH20 -- 10 µl     Total
 * Ligate the J01008-2-1 and 2 fragment and R0010 vector
 * 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.