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" : "2013-07-31",
-	"author" : "thomas",
-	"title" : "pSpac + GFP cloning!!!",
-	"content" : "<html>Today I started a new cloning in order to obtain GFP in the vector pSBBs0K-Pspac (<a href=\"http://parts.igem.org/Part:BBa_K823026\">BBa_K823026</a>). This construct will be useful to Emil in order to characterize Pspac at difference concentration of inducer (IPTG). I started performing a PCR on the GFP following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Phusion-PCR\">Phusion PCR protocol</a>. I used BB_fwd and BB_rev primers to obtain an amplification of the insert. This because the GFP backbone (<a href=\"http://parts.igem.org/partsdb/get_part.cgi?part=BBa_E0840\">BBa_E0840</a>) had the same antibiotic resisistance of our destination vector BBa_K823026 (Amp). I obtain a PCR product with a concentration of 146 ng/&micro;l, confirmed by electrophoresis analysis.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel image|<html><center><img src=\"https://static.igem.org/mediawiki/2013/5/5c/Tn-2013_gel_E0840_PCR.jpg\" width=\"450px\" /></center></html>}}<html>  I continued then with the restriction digestions following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">this protocol</a>. I digested BBa_K823026 using SpeI and PstI, and PCR product with XbaI and PstI. <br/>The two digestion products were then purified and quantified: GFP PCR 60 ng/&micro;l, BBa_K823026 19 ng/&micro;l. The last step was then the ligation that was performed following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Ligation\">this protocol</a>. The ligation products were then plated on CM plates. </html>",
-	"tags" : "pSpac-GFP"
-}