Team:UNITN-Trento/Notebook/Labposts/06/27

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Revision as of 07:41, 9 September 2013

{ "date" : "2013-06-17", "author" : "gabriele-emil", "title" : "SAMsynthetase: like the legend of the phoenix,'all ends with beginning", "content" : "Today we started anew extracting SAMsynthetase from the genome of E. coli strain MG1655, following the usual protocol (2 samples for Gabriele and 2 for Emil). Then we performed an electrophoresis on a 1% agarose gel.

Gel Result

Loading scheme
G1	G2	1kb ladder	E1	E2

Sadly, something went wrong with G2 sample (probably Gabriele forgot to add something to the PCR mix). But the gel is very beautiful!!!

Then G1, E1 and E2 samples were purified using Wizard® SV Gel and PCRClean-Up System and then quantified using the Nanodrop.

Quantification results

Sample	Quantity
G1	80.2ng/µl
E1	65.7ng/µl
E2	60ng/µl

Sadly, the quantities were not so high, but the results was good anyway!

Finally we prepared overnight digestion of SAMsynthetase (G1 and E1, E2 was put at -20°C) and pSB1C3 linearized both with XbaI and PstI-HF, using the digestion protocol. We used Nebuffer4 and the mix were prepared as follows:

Digestion mix

	G1	E1	linear pSB1C3
Nebuffer4	10µl	5µl
XbaI	1µl
PstI-HF	1µl
BSA	1µl [from 100X stock]	5µl [from 10X stock]
DNA [3µg]	37.41µl	45.66µl	37.31µl
Water	49.59µl	41.34µl	0.69µl
Total	100µl	50µl

The mix were incubated at 37°C overnight.", "tags" : "SAMsynthetase" }