Team:Paris/August 13
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(→Minipreps: Plasmid extraction) |
(→PCR screening) |
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|L100.1 | |L100.1 | ||
| rowspan="3"| rbs TetR - ECFP<br>D110 (BV) - D130 (BI) | | rowspan="3"| rbs TetR - ECFP<br>D110 (BV) - D130 (BI) | ||
- | | rowspan="3"| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]] | + | | rowspan="3"| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]] |
|- | |- | ||
|S146.2 | |S146.2 | ||
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|L101.1 | |L101.1 | ||
| rowspan="3"| rbs TetR - GFP tripart<br>D110 (BV) - D131 (BI) | | rowspan="3"| rbs TetR - GFP tripart<br>D110 (BV) - D131 (BI) | ||
- | | rowspan="3"| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]] | + | | rowspan="3"| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]] |
|- | |- | ||
|S147.2 | |S147.2 | ||
Line 222: | Line 222: | ||
|L120.1 | |L120.1 | ||
| rowspan="3"| tetR repressible promoter - ECFP<br>D106 (BV) - D130 (BI) | | rowspan="3"| tetR repressible promoter - ECFP<br>D106 (BV) - D130 (BI) | ||
- | | rowspan="3"| [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]] | + | | rowspan="3"| [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]] |
|- | |- | ||
|S149.2 | |S149.2 | ||
Line 233: | Line 233: | ||
|L122.1 | |L122.1 | ||
| RBS-lasI - ECFP <br>D107 (BV) - D130 (BI) | | RBS-lasI - ECFP <br>D107 (BV) - D130 (BI) | ||
- | | [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]] | + | | [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]] |
|- | |- | ||
|S151.1 | |S151.1 | ||
|L123.1 | |L123.1 | ||
| rowspan="3"| RBS lasI - ECFP<br>D107 (BV) - D131 (BI) | | rowspan="3"| RBS lasI - ECFP<br>D107 (BV) - D131 (BI) | ||
- | | rowspan="3"| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]] | + | | rowspan="3"| [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]] |
|- | |- | ||
|S151.2 | |S151.2 | ||
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==> results: PCR products can be purified using the Qiaquick Gel Extraction kit. We just have to replace the buffer PBI by the buffer QG! | ==> results: PCR products can be purified using the Qiaquick Gel Extraction kit. We just have to replace the buffer PBI by the buffer QG! | ||
- | ==PCR screening== | + | == '''PCR screening'''== |
+ | |||
+ | === Electrophoresis Setting === | ||
4 more transformants of L130 (pFlhB into J61002) are screened by PCR. | 4 more transformants of L130 (pFlhB into J61002) are screened by PCR. | ||
- | *PCR screening programm | + | *PCR screening programm; elogation time: 1 min 30 |
- | + | ||
*template: colonies from the transformation plate | *template: colonies from the transformation plate | ||
*positive control: S142 (J61002) | *positive control: S142 (J61002) | ||
*negative control: no template | *negative control: no template | ||
*primers: O18 and O19 | *primers: O18 and O19 | ||
- | ===Electrophoresis=== | + | |
+ | |||
+ | ===Results of Electrophoresis=== | ||
[[Image:Screening-PCR-080813.JPG|thumb|'''Gel 1''': PCR screening ('''2-7''') & PCR product purification using the Qiaquick Gel Extraction kit ('''8''')]] | [[Image:Screening-PCR-080813.JPG|thumb|'''Gel 1''': PCR screening ('''2-7''') & PCR product purification using the Qiaquick Gel Extraction kit ('''8''')]] | ||
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*10 µL loaded | *10 µL loaded | ||
- | {|border="1" | + | {| style="text-align: center;" border="1" |
- | + | |'''well n°''' | |
- | + | |1 | |
- | + | |2 | |
- | + | |3 | |
- | + | |4 | |
- | + | |5 | |
- | + | |6 | |
- | + | |7 | |
- | + | |8 | |
|- | |- | ||
- | + | |'''sample''' | |
- | + | |100 bp DNA ladder | |
- | + | |positive control | |
- | + | |negative control | |
- | + | |'''L130.3''' | |
- | + | |'''L130.4''' | |
- | + | |'''L130.5''' | |
- | + | |'''L130.6''' | |
- | + | |PCR product (L130.8) purified by the Qiaquick Gel Extraction kit | |
|- | |- | ||
- | + | |'''red fluorescence''' | |
|rowspan="3"| | |rowspan="3"| | ||
- | + | |strain a little bit pink | |
- | + | |not concerned | |
- | + | |no | |
- | + | |no | |
- | + | |no | |
- | + | |no | |
- | |rowspan="3"| | + | |rowspan="3"|not concerned |
|- | |- | ||
- | + | |'''expected size''' | |
- | + | |1,161 kb | |
- | + | |0 kb | |
|colspan="4"|<center>'''1,338 kb'''</center> | |colspan="4"|<center>'''1,338 kb'''</center> | ||
|- | |- | ||
- | + | |'''measured size''' | |
- | | | + | |style="background: #cbff7B"| 1,2 kb |
- | | | + | |style="background: #cbff7B"| 0 kb |
- | | | + | |style="background: #ff6d73"| 1,2 kb |
- | | | + | |style="background: #ff6d73"| 1,2 kb |
- | | | + | |style="background: #ff6d73"| 1,2 kb |
- | | | + | |style="background: #ff6d73"| 1,2 kb |
|} | |} | ||
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=='''Transformation of the ligation we did [[Team:Paris/August_12|yesterday]]'''== | =='''Transformation of the ligation we did [[Team:Paris/August_12|yesterday]]'''== | ||
We transformed L 139, L140, L141 and L142 following the [[Team:Paris/Notebook/Protocols#Transformation|standard protocol using Invitrogen's TOP 10 chemically competent cells]]. The positive control is a transformation with pUC19 and the negative control has no plasmid. | We transformed L 139, L140, L141 and L142 following the [[Team:Paris/Notebook/Protocols#Transformation|standard protocol using Invitrogen's TOP 10 chemically competent cells]]. The positive control is a transformation with pUC19 and the negative control has no plasmid. | ||
- | |||
- |
Latest revision as of 16:12, 15 August 2008
Sequencing Minipreps we did yesterday
Minipreps: Plasmid extraction
Glycerol Stocks
Assay to purify a PCR product using the Qiaquick Gel Extraction kit
=> see Gel 1, well n° 8 ==> results: PCR products can be purified using the Qiaquick Gel Extraction kit. We just have to replace the buffer PBI by the buffer QG! PCR screeningElectrophoresis Setting4 more transformants of L130 (pFlhB into J61002) are screened by PCR.
Results of Electrophoresis
==> The L130 transformants analysed are not correct. Transformation of the ligation we did yesterdayWe transformed L 139, L140, L141 and L142 following the standard protocol using Invitrogen's TOP 10 chemically competent cells. The positive control is a transformation with pUC19 and the negative control has no plasmid. |