IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-20
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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
- Transformation test
Competent Cell/plasmid transformation growth 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
- 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:
- 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
- Digesting all plasmids above, R0010, E0040.B0015 and E0040 with EcoRI/PstI.
- Each digestion system contains:
1 µl 10*H buffer 0.25 µl EcoRI 0.25 µl PstI 3 µl Plasmid 5.5 µl ddH20 -------------------------- 10 µl Total
- 37℃ culutre for 3 hours.
- from left to right:
- R0010<-J01008-1 @ EcoRI/PstI
- R0010<-J01008-2 @ EcoRI/PstI
- R0010<-J01008-3 @ EcoRI/PstI
- R0010<-J01008-4 @ EcoRI/PstI
- R0010<-J01008-5 @ EcoRI/PstI
- R0010<-J01008-6 @ EcoRI/PstI
- R0010 @ EcoRI/PstI
- R0040.J01010->E0040.B0015-1 @ EcoRI/PstI
- R0040.J01010->E0040.B0015-2 @ EcoRI/PstI
- R0040.J01010->E0040.B0015-3 @ EcoRI/PstI
- E0040.B0015 @ EcoRI/PstI
- E0040 @ EcoRI/PstI
- marker (DL2000 Plus)
- Conclusion:
- R0040.J01010.E0040.B0015 makes it.
- R0010<-J01008 fails.
- Stripe the R0040.J01010.E0040.B0015-2 on Kan+ LB plate for storage.
Sequencing
- Send the following precultures to Invitrogen for 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
- Use vf2 and vr primer for pSB plasmids and T7 primer for T3 plasmids.
Ligation: R0010<-J01008-2-1,2
- Ligate the J01008-2-1 and 2 fragment and R0010 vector
- Ligation system contains:
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
- 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.