Team:UNITN-Trento/Notebook/Labposts/07/32

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Latest revision as of 10:39, 3 October 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"}

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+{"date" : "2013-07-15","author" : "gabriele","title" : "Let's try again","content" : "<html><h3>What happened to the inocula???</h3>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 :(<br/>I miniprepped the only not-red inoculum, the \"1:1 A\" which had a concentration of 230.5ng/&micro;l. Then I stocked the sample at -20&deg;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 a&Delta;length higher than 500bp (otherwise it is impossible to distinguish the two bands). Then enzyme that we will use is AgeI.<br/><hr><h3>Another digestion</h3>So, I purified the SAM synthetase extracted through PCR on <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-11-gabriele\">11/07</a>.<center><table class=\"tn-sp-table\"><tr><th>sample</th><th>Quantity</th></tr><tr><td>G1</td><td>116.7ng/&micro;l</td></tr><tr><td>G2</td><td>62.6ng/&micro;l</td></tr><tr><td>G3</td><td>82ng/&micro;l</td></tr></table></center>Then I added 1&micro;l of DpnI to the linear pSB1C3 sample (<b>G2A</b> from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-01-Gabriele\">01/07</a>) and incubated it at 37&deg;C for 1 hour. Then I prepared an overnight digestion following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">usual protocol</a>.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Digestion mixes|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>EX-SAMsynth-SP</th><th>linear pSB1C3</th></tr><tr><td>Template</td><td>34.27&micro;l</td><td>50&micro;l</td></tr><tr><td>EcoRI-HF</td><td rowspan=\"2\">2.5&micro;l</td><td rowspan=\"2\">1.5&micro;l</td></tr><tr><td>PstI-HF</td></tr><tr><td>NEBuffer 2</td><td rowspan=\"2\">10&micro;l</td><td rowspan=\"2\">5&micro;l</td></tr><tr><td>BSA</td></tr><tr><td>Water</td><td>40.73&micro;l</td><td>0</td></tr></table></center></html>}}<html></html>","tags" : "SAMsynthetase"}
-	"date" : "2013-07-17",
-	"author" : "fabio-bruno",
-	"title" : " blue and red light sensors!!",
-	"content" : "<html>  today we made 2 inocula for each one of the 4 parts we transformed!! We’re ready to see what’s next! </html>",
-	"tags" : " YF1_FixJ - FixK2- ho1_PcyA_cph8-OmpR"
-}