Team:UNITN-Trento/Notebook/Labposts/07/08

From 2013.igem.org

(Difference between revisions)

Revision as of 07:21, 9 September 2013

{ "date" : "2013-07-15", "author" : "gabriele", "title" : "Let's try again", "content" : "

What happened to the inocula???

First, I wanted to miniprep 3 inocula... but two were RED!!! So sad, the RFP was present... that's because I forgot to treat the linear plasmid with DpnI :(
I miniprepped the only not-red inoculum, the \"1:1 A\" which had a concentration of 230.5ng/µl. Then I stocked the sample at -20°C. The possible presence of Plac+RFP is a problem since it is nearly as long as the SAM synthetase gene. So, for the screening, I need an enzyme that cuts only one (either SAM synthetase OR Plac+RFP) forming two fragment with aΔlength higher than 500bp (otherwise it is impossible to distinguish the two bands). Then enzyme that we will use is AgeI.

Another digestion

So, I purified the SAM synthetase extracted through PCR on 11/07.

sample	Quantity
G1	116.7ng/µl
G2	62.6ng/µl
G3	82ng/µl

Then I added 1µl of DpnI to the linear pSB1C3 sample (G2A from 01/07) and incubated it at 37°C for 1 hour. Then I prepared an overnight digestion following the usual protocol.

Digestion mixes

	EX-SAMsynth-SP	linear pSB1C3
Template	34.27µl	50µl
EcoRI-HF	2.5µl	1.5µl
PstI-HF
NEBuffer 2	10µl	5µl
BSA
Water	40.73µl	0

", "tags" : "SAMsynthetase" }