Team:UNITN-Trento/Notebook/Labposts/07/57

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Revision as of 07:55, 9 September 2013

{ "date" : "2013-07-11", "author" : "gabriele", "title" : "Extraction (again) and ligation", "content" : "

SAM synthetase extraction

Last time I determined the correct protocol to extract SAM synthetase from the E. coli using the new plasmid (at the end, the protocol was the usual one). I performed three PCRs (G1, G2, G3) and run the products on 1% agarose gel.

Gel

Loading scheme
1kb ladder	G1	G2	G3

$\"Gel\"$

Ligation

At first, I added 1µl of SAP to pSB1C3 O/N digestion and 1µl of DpnI to SAMsynthetase O/N digestion, and incubated the samples at 37°C for 1.5h. Then I quantified the digestions.

Sample	Quantity
SAM synthetase	12.2ng/µl
pSB1C3	14.0ng/µl

Then I performed the ligation and left the reaction run for 2h at room temperature.

Ligation Mixes

	CTRL	1:1	1:2
Buffer	4µl
Plasmid	14.29µl
Insert	0	9.15µl	18.3µl
Ligase	1µl
Water	20.71µl	11.56µl	2.41µl

Then I transformed the ligation products in NEB10β", "tags" : "SAMsynthetase" }