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Revision as of 14:53, 31 July 2007 (view source) Davidpz (Talk | contribs) (→PCR mutagenesis DGAT) ← Older edit		Revision as of 15:40, 31 July 2007 (view source) David.bikard (Talk | contribs) Newer edit →
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		+	== pTOPO cloning ==
		+	Cloning of PCR  products: P5, P6, P7, P8, P9, P10 (see [[Paris/Freezer]])
		+
		+
		+	== Transformation ==
		+
		+	Ligations from yesterday
		+
		+
		+
	== PCR mutagenesis DGAT ==		== PCR mutagenesis DGAT ==

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Revision as of 15:40, 31 July 2007

pTOPO cloning

Cloning of PCR products: P5, P6, P7, P8, P9, P10 (see Paris/Freezer)

Transformation

Ligations from yesterday

PCR mutagenesis DGAT

DGAT has Pst1 restriction site. We want to mutagenize it to do a biobrick.

PCR : DGAT1
PCR Settings		Buffer (5x)	5x 10µL			Expected size
Annealing (°C)		MgCl2 10µM	10µM 0µL			1087
50		dNTP 10µM	10µM 1µL			Success
Time Elongation		Oligo F 10µM	27 ForDGAT1	2.5µL		YES
2m00'		Oligo R 10µM	13 DPst1-DGAT-R	2.5µL		Image (click to enlarge)
Number cycles		Water	33µl
35		Polymerase	Phusion 0.5µL			Band (0=ladder)
		DNA	Miniprep pKS::DGAT (1µL = 138ng)			3, 4, 5, 6 (11 = control without DNA matrix)

PCR : DGAT2
PCR Settings		Buffer (5x)	5x 10µL			Expected size
Annealing (°C)		MgCl2 10µM	10µM 0µL			396
50		dNTP 10µM	10µM 1µL			Success
Time Elongation		Oligo F 10µM	12 DPst1-DGAT	2.5µL		YES
2m00'		Oligo R 10µM	27 Rev-DGAT1	2.5µL		Image (click to enlarge)
Number cycles		Water	33µl
35		Polymerase	Phusion 0.5µL			Band (0=ladder)
		DNA	Miniprep pKS::DGAT (1µL = 138ng)			7,8,9,10 (11 = control without DNA matrix)