"
Team:UNITN-Trento/Notebook/Labposts/07/27
From 2013.igem.org
(Difference between revisions)
| Line 1: | Line 1: | ||
{ | { | ||
| - | "date" : "2013-07- | + | "date" : "2013-07-15", |
| - | "author" : " | + | "author" : "gabriele", |
| - | "title" : " | + | "title" : "Let's try again", |
| - | "content" : "<html> | + | "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/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.<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/µl</td></tr><tr><td>G2</td><td>62.6ng/µl</td></tr><tr><td>G3</td><td>82ng/µl</td></tr></table></center>Then I added 1µl 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°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>34.27µl</td><td>50µl</td></tr><tr><td>EcoRI-HF</td><td rowspan=\"2\">2.5µl</td><td rowspan=\"2\">1.5µl</td></tr><tr><td>PstI-HF</td></tr><tr><td>NEBuffer 2</td><td rowspan=\"2\">10µl</td><td rowspan=\"2\">5µl</td></tr><tr><td>BSA</td></tr><tr><td>Water</td><td>40.73µl</td><td>0</td></tr></table></center></html>}}<html></html>", |
| - | "tags" : " | + | "tags" : "SAMsynthetase" |
} | } | ||
Revision as of 08:30, 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 |
Digestion mixes
",
"tags" : "SAMsynthetase"
}
| 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 |
