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

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(Created page with "{ "date" : "2013-06-17", "author" : "gabriele-emil", "title" : "SAMsynthetase: ''like the legend of the phoenix,''''all ends with beginning''", "content" : "<html>Today we st...")

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
G1G21kb ladderE1E2


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
SampleQuantity
G180.2ng/µl
E165.7ng/µl
E260ng/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
G1E1linear pSB1C3
Nebuffer410µl5µl
XbaI1µl
PstI-HF1µl
BSA1µl [from 100X stock]5µl [from 10X stock]
DNA [3µg]37.41µl45.66µl37.31µl
Water49.59µl41.34µl0.69µl
Total100µl50µl
The mix were incubated at 37°C overnight.", "tags" : "SAMsynthetase" }