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Team:Warsaw/Calendar-Main/11 July 2009
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Ffijalkowski (Talk | contribs) (→Creating devices to test promoters in E. coli strains (devices BBa_K177024 and BBa_K177025)) |
Ffijalkowski (Talk | contribs) (→Creating devices to test promoters in E. coli strains (devices BBa_K177024 and BBa_K177025)) |
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====Franek lub Monika opisze minilizy dla Moniki ==== | ====Franek lub Monika opisze minilizy dla Moniki ==== | ||
Revision as of 00:29, 12 July 2009
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Gel out phoP/phoQ
Kama
Tasks:
- Isolation of fragment of the correct lenght(˜2200bp)from the gel was performed with the [http://aabiot.com/products/dna_purification/dna_fragments/gel_out/protocol_gel_out.pdf A&A "Gel-out" kit].
Construction of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 K177012] operon1_part2
Ania
Tasks:
- Transformation of chemocompetent strain of E. with [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid taken out of the distribution 200 Kit Plate 1 well 2O.
- Alkaline lysis of bacterial cultures to obtain plasmids containing following bricks:
[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0051 BBa_R0051] - promoter (lambda cI regulated); [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032] - RBS.3 (medium)
- Digestion to confirm plasmid extraction. DNA that should contain R0051 on pSB1A2 plasmid was digested with PvuI and HindII. Digestion mix contained 5µl of extracted DNA, 2µl of Fermentas BamHI buffer , 0.3µl of each enzyme and water added to obtain 20µl total volume.
| DNA sample | restriction enzymes | expected fragments [bp] |
|---|---|---|
| R0051(pcI) on pSB1A2 | PvuI HindII | 695, 1447 |
Creating devices to test promoters in E. coli strains (devices [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025])
Franek
Tasks:
- Alkaline lysis of bacterial cultures to obtain plasmids containing [http://partsregistry.org/Part:BBa_R0010 BBa_R0010 - lacI regulated promoter] and [http://partsregistry.org/Part:BBa_R0080 BBa_R0080 - AraC regulated promoter] bricks
- Digestion to confirm plasmid extraction.
Methods:
- 3 test tubes with 5ml LB and ampicillin were first inoculated with colonies containing [http://partsregistry.org/Part:BBa_R0010 placI] or [http://partsregistry.org/Part:BBa_R0080 pAraC] bricks on [http://partsregistry.org/Part:pSB1A2 pSB1A2] plasmid. The cultures were incubated over night at 37°C. Alkaline lysis was performed on both cultures, according to our standar procedure. The pellet from 5 ml of bacteria was used.
- DNA that should contain [http://partsregistry.org/Part:BBa_R0080 pAraC] on [http://partsregistry.org/Part:pSB1A2 pSB1A2] plasmid was digested with BamHI PvuI, [http://partsregistry.org/Part:BBa_R0010 placI] on [http://partsregistry.org/Part:pSB1A2 pSB1A2] was digested with IPvuI and PvuII . Digestion mix contained 5µl of extracted DNA, 2µl of Fermentas BamHI buffer , 0.3µl of each enzyme and water added to obtain 20µl total volume.
| DNA sample | restriction enzymes | expected fragments [bp] |
|---|---|---|
| [http://partsregistry.org/Part:BBa_R0010 placI on pSB1A2] | PvuI PvuII | 728, 1351 |
| [http://partsregistry.org/Part:BBa_R0080 pAraC on pSB1A2] | BamHI PvuI | 782, 1446 |
Results:
Franek lub Monika opisze minilizy dla Moniki
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