IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-22: Difference between revisions
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=Tandem oriT by Qu Mingzhi= | |||
==Amplification Culture of PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 (III)== | ==Amplification Culture of PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 (III)== | ||
*Amplification Culture of PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 from plates. | *Amplification Culture of PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 from plates. | ||
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*PlacI-E0240 plasmid | *PlacI-E0240 plasmid | ||
[[Image:Peking_2007-8-22_R0010gel_PlacI-I741051gel_E0240gel_PlacI-I741051plasmid_R0010plasmid.jpg ]] | [[Image:Peking_2007-8-22_R0010gel_PlacI-I741051gel_E0240gel_PlacI-I741051plasmid_R0010plasmid.jpg ]] | ||
=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 | |||
[[Image:Example.jpg]] | |||
*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: | |||
<pre> | |||
7.5 µl J01008 fragment | |||
1 µl R0010 vector | |||
0.5 µl Super T4-Ligase | |||
1 µl 10 X ligation buffer | |||
-------------------------- | |||
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. |
Revision as of 02:05, 31 August 2007
Tandem oriT by Qu Mingzhi
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.