Team:Paris/August 14

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Revision as of 12:38, 15 August 2008

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Results of the PCR of the wonderful promoter of fliL

Amplification of pfliL

Electrophoresis settings :

Gel 1.5% agar
Ladder 100 bp
Volume of template : 3 µL

Name	Promotor	Well	Expected size	Measured size
PCR_135	pfliL	2	197 bp	~ 200 bp
PCR_135'	Negative Control	3	0 bp	0 bp

Remarks:
We observe that the bands are curved, we suppose that the wells were not very clean.
The size of fliL is good, we will digest it and ligate it today.

Digestion and Ligation of the wonderful promoter of fliL

As the primer used to amplify the promoter of fliL had only two nucleotides after the restriction sites, we tried the two digestions possible : EcoRI + SpeI and XbaI + PstI.

Digestion

Protocol :

Name	Genes	Water	DNA	Buffer n°2 10X	BSA 100X	Enz 1	Enz 2
D149	fliL	23 µL	2 µl	3 µl	0.30µl	EcoRI	SpeI
D150	fliL	23 µL	2 µl	3 µl	0.30µl	XbaI	PstI
D137	pSB3K3	23 µL	2 µl	3 µl	0.30µl	EcoRI	SpeI
D152	pSB3K3	23 µL	2 µl	3 µl	0.30µl	XbaI	PstI

Incubate 2h30 at 37°C
20 min at 65°C to denaturate the enzymes.

Washing of the digestions

We washed the DNA following the standard protocol.

Ligation

Ligation name	Insert name	Volume of insert µL	Vector name	Volume of Vector µL
L 143	D 149 (pfliL)	2	D 137 (pSB3K3)	6
L 144	D 150 (pfliL)	2	D 152 (pSB3K3)	6

2 µL Ligase Buffer 10X
X µg/µL vector
Y µg/µL insert
Pure water qsp 20 µL
1 µL T4 ligase
O/N at 16°C

T1 and T2 are the autoligation controls for L143 and L144.

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 {{Paris/Calendar_Links|August 13|August 15}}
 =='''Results of the PCR of the wonderful promoter of ''fliL'' '''==
 [[Image:KR000147.jpg|thumb|Amplification of pfliL]]