IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-15: Difference between revisions
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==Amplification Culture of E0240== | ==Amplification Culture of E0240== | ||
*select Positive E0240 Colonies from Plate,Culture in liquid LB,waiting for mini-prep. | *select Positive E0240 Colonies from Plate,Culture in liquid LB,waiting for mini-prep. | ||
=Lock & Key By Yu Tao= | |||
==R0010.J01008 and R0040.J01010 Digestion Product Purification== | |||
*Use Transgen EasyPure PCR Purification Kit for R0010<-J01008 and Quick Gel Extraction Kit for R0040<--J01010. | |||
*30uL per tube after purflication, one tube, respectively. | |||
===Electrophorsis Result=== | |||
*from ''left'' to ''right'': | |||
#DL 2000 plus marker | |||
#R0010.J01008 @ SpeI/PstI purified digestion product | |||
#R0040.J01010 @ EcoRI/SpeI purified digestion product | |||
[[Image:Example.jpg]] | |||
==Ligation: R0010.J01008<-B0015 and R0040.J01010->E0040.B0015== | |||
===Recheck previous B0015 fragment and E0040.B0015 vector result=== | |||
*from ''left'' to ''right'': | |||
#B0015-1 @ XbaI/PstI purified digestion product | |||
#B0015-2 @ XbaI/PstI purified digestion product | |||
#E0040.B0015 @ EcoR/XbaI purified digestion product | |||
#DL2000 plus marker | |||
[[Image:Example.jpg]] | |||
*Conclusion: Use the B0015-2 @ XbaI/PstI purified digestion product and E0040.B0015 @ EcoR/XbaI purified digestion product. | |||
*Ligate the R0040.J01010 fragment and E0040.B0015 vector, as well as the B0015 fragment and R0010<-J01008 vector. | |||
*Ligation system contains: | |||
<pre> | |||
7 µl R0040.J01010 fragment / 3 µl B0015 fragment | |||
1 µl E0040.B0015 vector / 1 µl R0010<-J01008 vector | |||
0.5 µl Super T4-Ligase | |||
1 µl 10 X ligation buffer | |||
0 µl ddH20 / 4 µl ddH20 | |||
-------------------------- | |||
9.5 µl Total | |||
</pre> | |||
*The negative control group contains no fragment but ddH2O instead. | |||
*10min at 16℃. | |||
==Transformation: R0010.J01008<-B0015 and R0040.J01010->E0040.B0015== | |||
*Transform all ligation products into 100 µl DH5α competent cells. | |||
*Culture all R0010.J01008<-B0015 cells at Amp+ LB plate and all R0040.J01010->E0040.B0015 cells at Kan+ LB plate for 12 hours. | |||
*Result to be seen tomorrow. | |||
==New competent cells preparation== | |||
*Prepare Competent Cells III | |||
==Competent Cells III efficiency test== | |||
*Trasform 1uL R0010 plasmid into competent cells for test. | |||
<pre> | |||
| Competent Cells II | Competent Cells III | Competent Cells III | |||
------------------------------------------------------------------------------------------------ | |||
R0010 | 1 uL | 1 uL | 0 uL | |||
------------------------------------------------------------------------------------------------ | |||
ddH2O | 0 uL | 0 uL | 1 uL | |||
------------------------------------------------------------------------------------------------ | |||
antibiotics resistency test | Amp | Amp | Amp/Kan,respectively | |||
</pre> | |||
==Double Digestion: R0040.J01010== | |||
*Because the purified digestion product of R0040.J01010 is almost unseen in the electrophoresis result, I decide to redo it once more. | |||
*Digesting R0040.J01010 with EcoRI/SpeI. | |||
*Each digestion system contains: | |||
<pre> | |||
4 µl 10*H | |||
1 µl EcoRI | |||
1 µl SpeI | |||
25 µl Plasmid | |||
9 µl ddH20 | |||
-------------------------- | |||
40 µl Total | |||
</pre> | |||
*37℃ culutre overnight. |
Latest revision as of 20:00, 28 August 2007
Tandem OriT by Qu Mingzhi
transformation R-OriT & S-OriT
- Use pEASY-3 as cloning vector .(Amp+)
- tramsformation system contains :3uL R-OriT, S-OriT PCR product(after purflication), 1uL pEasy-T3
- the Culture Dish(LB/Amp+) are trated with 5uL 1M IPTG & 40uL 40mg/mL x-Gal.
- NEXT DAY: 10+ white cloney received.
Amplification Culture of E0240
- select Positive E0240 Colonies from Plate,Culture in liquid LB,waiting for mini-prep.
Lock & Key By Yu Tao
R0010.J01008 and R0040.J01010 Digestion Product Purification
- Use Transgen EasyPure PCR Purification Kit for R0010<-J01008 and Quick Gel Extraction Kit for R0040<--J01010.
- 30uL per tube after purflication, one tube, respectively.
Electrophorsis Result
- from left to right:
- DL 2000 plus marker
- R0010.J01008 @ SpeI/PstI purified digestion product
- R0040.J01010 @ EcoRI/SpeI purified digestion product
Ligation: R0010.J01008<-B0015 and R0040.J01010->E0040.B0015
Recheck previous B0015 fragment and E0040.B0015 vector result
- from left to right:
- B0015-1 @ XbaI/PstI purified digestion product
- B0015-2 @ XbaI/PstI purified digestion product
- E0040.B0015 @ EcoR/XbaI purified digestion product
- DL2000 plus marker
- Conclusion: Use the B0015-2 @ XbaI/PstI purified digestion product and E0040.B0015 @ EcoR/XbaI purified digestion product.
- Ligate the R0040.J01010 fragment and E0040.B0015 vector, as well as the B0015 fragment and R0010<-J01008 vector.
- Ligation system contains:
7 µl R0040.J01010 fragment / 3 µl B0015 fragment 1 µl E0040.B0015 vector / 1 µl R0010<-J01008 vector 0.5 µl Super T4-Ligase 1 µl 10 X ligation buffer 0 µl ddH20 / 4 µl ddH20 -------------------------- 9.5 µl Total
- The negative control group contains no fragment but ddH2O instead.
- 10min at 16℃.
Transformation: R0010.J01008<-B0015 and R0040.J01010->E0040.B0015
- Transform all ligation products into 100 µl DH5α competent cells.
- Culture all R0010.J01008<-B0015 cells at Amp+ LB plate and all R0040.J01010->E0040.B0015 cells at Kan+ LB plate for 12 hours.
- Result to be seen tomorrow.
New competent cells preparation
- Prepare Competent Cells III
Competent Cells III efficiency test
- Trasform 1uL R0010 plasmid into competent cells for test.
| Competent Cells II | Competent Cells III | Competent Cells III ------------------------------------------------------------------------------------------------ R0010 | 1 uL | 1 uL | 0 uL ------------------------------------------------------------------------------------------------ ddH2O | 0 uL | 0 uL | 1 uL ------------------------------------------------------------------------------------------------ antibiotics resistency test | Amp | Amp | Amp/Kan,respectively
Double Digestion: R0040.J01010
- Because the purified digestion product of R0040.J01010 is almost unseen in the electrophoresis result, I decide to redo it once more.
- Digesting R0040.J01010 with EcoRI/SpeI.
- Each digestion system contains:
4 µl 10*H 1 µl EcoRI 1 µl SpeI 25 µl Plasmid 9 µl ddH20 -------------------------- 40 µl Total
- 37℃ culutre overnight.