Team:Paris/August 21
From 2008.igem.org
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(→Measurement of the concentration of D166 & D167 purified) |
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{{Paris/Calendar_Links|August 20|August 22}} | {{Paris/Calendar_Links|August 20|August 22}} | ||
+ | =''' Cloning of FlhB promoter'''= | ||
+ | |||
+ | ==Protocol== | ||
+ | We used the Phusion polymerase to amplify this promoter. | ||
+ | |||
+ | * '''Preparation of the template''' : | ||
+ | Resuspension of 1 colony ''E.coli'' K12 strain MG 1655 in 100µl of water. | ||
+ | |||
+ | * '''Preparation of PCR mix''' : | ||
+ | <br> 1µl of dNTP | ||
+ | <br> 10µl Buffer Phusion 5X | ||
+ | <br> 2.5µl O 108 | ||
+ | <br> 2.5µl O 109 | ||
+ | <br> 1µl Template | ||
+ | <br> 1µl Phusion | ||
+ | <br> 33µL pure water | ||
+ | |||
+ | |||
+ | ==Result== | ||
+ | [[Image:KR00019b.jpg| thumb| Vérification of pFlhB] | ||
+ | |||
+ | {|border="1" style="text-align: center" | ||
+ | |'''Well''' | ||
+ | |1 | ||
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+ | |- | ||
+ | |'''Sample''' | ||
+ | |100 bp ladder | ||
+ | | | ||
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+ | | | ||
+ | | | ||
+ | |- | ||
+ | |'''Expected size (pb)''' | ||
+ | | | ||
+ | | | ||
+ | | | ||
+ | | | ||
+ | | | ||
+ | | | ||
+ | | | ||
+ | | | ||
+ | |- | ||
+ | |'''Measured size (pb)''' | ||
+ | | | ||
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+ | | | ||
+ | | | ||
+ | | | ||
+ | |} | ||
+ | |||
+ | |||
+ | |||
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='''Construction of pFlgA - YFP tripart (+/- LVA)'''= | ='''Construction of pFlgA - YFP tripart (+/- LVA)'''= | ||
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='''Screening of the cloning of E0240 and FlhDC+promotor'''= | ='''Screening of the cloning of E0240 and FlhDC+promotor'''= |
Revision as of 20:06, 21 August 2008
Cloning of FlhB promoterProtocolWe used the Phusion polymerase to amplify this promoter.
Resuspension of 1 colony E.coli K12 strain MG 1655 in 100µl of water.
Result[[Image:KR00019b.jpg| thumb| Vérification of pFlhB]
Construction of pFlgA - YFP tripart (+/- LVA)Aim : Construction of "pFlgA-RBS-YFP-dbl ter" (pFlgA-E0430/E0432) DigestionGel ExtractionProtocol [[Image:KR00019b.jpg| thumb|Gel Extraction of D166-D167]
Measurement of the concentration of D166 & D167 purifiedProtocol (it's same that for Miniprep)
Ligation
Screening of the cloning of E0240 and FlhDC+promotorWe obtained single colonies with the dilution 100.
PCR screeningreaction mixture (25 µL)
PCR screening programm
Electrophoresis
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