From 2013.igem.org
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| | { | | { |
| - | "date" : "2013-07-15", | + | "date" : "2013-07-03", |
| - | "author" : "gabriele", | + | "author" : "fabio-viola", |
| - | "title" : "Let's try again", | + | "title" : "BACILLUS SUBTILIS COMES BACK TO LIFE!", |
| - | "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>", | + | "content" : "<html> HORROR!! We lost kind of the whole bacillus that we had, during the night, cause the liquid cultures were disrupted in the shaker at 26!! Luckily we have a survivor, the liquid culture at 37, and all the plates. <br>From the survivor we made further liquid cultures ( at 26°, 1:50 and 1:100; at 37° 1:50 and 1:100) <br>As soon as they became very cloudy we went along with the glycerol stock preparation and put our bacillus at -80°. The one that weren’t cloudy have been kept in the shaker all night long. <br>In the afternoon we inoculated some colonies from the plates in 5 ml of nutrient broth +starch.</html>", |
| - | "tags" : "SAMsynthetase" | + | "tags" : " Bacillus subtilis" |
| | } | | } |
Revision as of 08:23, 3 October 2013
{
"date" : "2013-07-03",
"author" : "fabio-viola",
"title" : "BACILLUS SUBTILIS COMES BACK TO LIFE!",
"content" : " HORROR!! We lost kind of the whole bacillus that we had, during the night, cause the liquid cultures were disrupted in the shaker at 26!! Luckily we have a survivor, the liquid culture at 37, and all the plates.
From the survivor we made further liquid cultures ( at 26°, 1:50 and 1:100; at 37° 1:50 and 1:100)
As soon as they became very cloudy we went along with the glycerol stock preparation and put our bacillus at -80°. The one that weren’t cloudy have been kept in the shaker all night long.
In the afternoon we inoculated some colonies from the plates in 5 ml of nutrient broth +starch.",
"tags" : " Bacillus subtilis"
}
"
