From 2013.igem.org
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| - | { | + | {"date" : "2013-07-11","author" : "emil","title" : "The incompetent Bacillus subtilis saga:bacillus medium begins","content" : "<html>Firstable I did an inoculum of Neb10β in order to make them competent the next day.I started to prepare the media needed for the transformation of Bacillus according to LMU munich protocol but due to the failure of this protocol I won't report the protocol.</html>","tags" : "Bacillus subtilis"} |
| - | "date" : "2013-07-15",
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| - | "author" : "gabriele",
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| - | "title" : "Let's try again",
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| - | "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>",
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| - | "tags" : "SAMsynthetase"
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| - | } | + | |
Latest revision as of 10:38, 3 October 2013
{"date" : "2013-07-11","author" : "emil","title" : "The incompetent Bacillus subtilis saga:bacillus medium begins","content" : "Firstable I did an inoculum of Neb10β in order to make them competent the next day.I started to prepare the media needed for the transformation of Bacillus according to LMU munich protocol but due to the failure of this protocol I won't report the protocol.","tags" : "Bacillus subtilis"}
"
