IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-20: Difference between revisions
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==Preparation of Competent Cells: F+, R751== | ==Preparation of Competent Cells: F+, R751== | ||
*Preparation F+, R751 competent cell for Conjugation test. | *Preparation F+, R751 competent cell for Conjugation test. | ||
===Comptent Cells efficiency test=== | |||
*Transformation test | |||
<pre> | |||
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+ | |||
</pre> | |||
==mini-prep PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 (II) == | ==mini-prep PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 (II) == | ||
*last mini-prep digesting test didn't show the correct backbone. | *last mini-prep digesting test didn't show the correct backbone. | ||
Line 23: | Line 37: | ||
====electrophoresis result==== | ====electrophoresis result==== | ||
* from ''left'' to ''right'': | * from ''left'' to ''right'': | ||
**1 | **1 Marker(DL2000 plus) | ||
**2-3 PlacI-E0240 @ EcoRI/PstI | **2-3 PlacI-E0240 @ EcoRI/PstI | ||
**4-5 PlacI-I741051-E0240 @ EcoRI/PstI | **4-5 PlacI-I741051-E0240 @ EcoRI/PstI | ||
[[Image:Peking_2007-8-20_PlacI-E0240_PlacI-I741051-E0240.jpg ]] | [[Image:Peking_2007-8-20_PlacI-E0240_PlacI-I741051-E0240.jpg ]] | ||
=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: | |||
<pre> | |||
1 µl 10*H buffer | |||
0.25 µl EcoRI | |||
0.25 µl PstI | |||
3 µl Plasmid | |||
5.5 µl ddH20 | |||
-------------------------- | |||
10 µl Total | |||
</pre> | |||
*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) | |||
[[Image:Example.jpg]] | |||
*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: | |||
<pre> | |||
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 | |||
</pre> | |||
*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. |
Latest revision as of 22:26, 30 August 2007
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.