Team:Paris/August 12
From 2008.igem.org
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- | ==Digestion of the PCR we did yesterday== | + | =='''Digestion and ligation of the PCR we did yesterday'''== |
Yesterday we amplified ''flhD'', ''flhC'', ''flhDC'' with its promoter and ''E040 RBS+'' | Yesterday we amplified ''flhD'', ''flhC'', ''flhDC'' with its promoter and ''E040 RBS+'' | ||
Before the digestion, we have to determine the DNA concentration of the templates. | Before the digestion, we have to determine the DNA concentration of the templates. | ||
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The DNA purification after gel extraction was done according the [[Team:Paris/Notebook/Protocols#Purification_.28Kit_Promega.29|standard protocol]]. | The DNA purification after gel extraction was done according the [[Team:Paris/Notebook/Protocols#Purification_.28Kit_Promega.29|standard protocol]]. | ||
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== Minipreps: Plasmid extraction== | == Minipreps: Plasmid extraction== |
Revision as of 14:57, 14 August 2008
Digestion and ligation of the PCR we did yesterdayYesterday we amplified flhD, flhC, flhDC with its promoter and E040 RBS+ Before the digestion, we have to determine the DNA concentration of the templates. Measurement of DNA concentrationWe used the biophotometer. We put 5 µL of template DNA in 95 µL of water. The Blank was made with 5 µL of EB Buffer (the one that was used for the elution of the DNA) in 95 µL of water.
DigestionProtocol
Results of the digestionFor the digestion after PCR, an electrophoresis is useless : we can not separate DNA fragments that have 10 bp of difference. LigationMinipreps: Plasmid extraction
Glycerol Stocks
Results of the transformations we did yesterdayYour results please Cyprien!!!??? Concentration of the MiniPreps
New PCR screening with the right primersTransformants with pFlgA, pFlgB and pFlhB cloned into J61002 are analysed by PCR but this time with the right primers: VF2 (O18) and VR (O19).
Electrophoresis
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