From 2013.igem.org
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| - | "date" : "2013-07-02", | + | "date" : "2013-07-23", |
| - | "author" : "gabriele", | + | "author" : "caterina", |
| - | "title" : "Another busy day", | + | "title" : "Trasformation of SAM", |
| - | "content" : "<html> <h3>SAM synthetase ligation in (linear) pSB1C3</h3>First of all, early in the morning I added 1µl of DpnI to the SAMsynthetase#G2 O/N digestion and 1µl of SAP to the linear_pSB1C3#G2 digestion and then incubated them at 37°C for 1.5 hours (afterward I inhibited the enzyme with 20 minutes at 80°C).<br/><br/>Then, I purified and quantified the two digestion samples:<center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantity</th></tr><tr><td>G1 <i>SAMsynthetase </i></td><td>18.8ng/µl</td></tr><tr><td>G2 <i>linear pSB1C3 </i></td><td>6. 5ng/µl</td></tr></table></center><br/ >After that, I performed the ligation (the plasmid had a too low concentration).<center><table class=\"tn-sp-table\"><tr><td style=\"border:none\"></td><th>Ctrl</th><th>0.5:1</th><th>0.5:2</th></tr><tr><td>Buffer</td><td colspan=\"3\">3µl</td></tr><tr><td>Plasmid</td><td colspan=\"3\">14.3µl</td></tr><tr><td>Insert</td><td>0</td><td>6µl</td><td>12µl</td></tr><tr><td>Ligase</td><td colspan=\"3\">1µl</td></tr><tr><td>Water</td><td>11.7µl</td><td>5.7µl</td><td>0µl</td></tr></table></center><br/> And, \"finally\", I transformed the ligations in NEB10β cells.<br/><br/><hr><h3>(2) SAMsynthetase amplification</h3>I amplified SAMsynthetase from the E2 sample (that was purified <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07- 01-Gabriele\">yesterday</a>) following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#SAM-extraction-genome-coli\">usual protocol</a>. The PCR was performed in triplicates.<br/><br/>The PCR products were run on a 1% agarose gel.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"5\">Loading scheme</th></tr><tr><td>E1A</td><td>E1B</td><td>E1C</td><td><i>empty</i></td><td>1kb ladder</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/6/6e/Tn-20130702-GG_SAMsynthetase_linearpSB1C3.jpg\" alt=\"Gel\" width=\"450px\"></center></html>}}<html><br/>As shown in the gel, a band at nearly 1200bp is present, confirming the success of the PCR.<br/><br/><hr><h3>(3) R0010 amplification</h3>I also amplified R0010 from <b>A</b> sample (R0010, 243.7ng/µl) from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-06-27-gabriele- emil\"> 27/ 06</a> . Being the first time amplifying this sequence, I performed (in triplicates or duplicates) a Phusion PCR using as primers the prefix Fw (Tm = 86& deg;C) and the suffix Rv (Tm = 90° C). <br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|PCR Mixes|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>Mix HF (x3)</th><th>Mix GC (x2)</th></tr><tr><td>Phusion GC buffer</td><td>0</td><td>10µl</td></tr><tr><td>Phusion HF buffer</td><td>10µl</td><td>0</td></tr><tr><td>dNTPs</td><td colspan=\"2\">1µl</td></tr><tr><td>primer Fw</td><td colspan=\"2\">2.5µl</td></tr><tr><td>primer Rv</td><td colspan=\"2\">2.5µl</td></tr><tr><td>template (50ng/µl)</td><td colspan=\"2\">0.5µl</td></tr><tr><td>Phusion pol</td><td colspan=\"2\">0.5µl</td></tr><tr><td>Water</td><td colspan=\"2\">33µl</td></tr></table></center></html>}}<html><br/>Given that R0010 is 200bp long, the PCR program was the following:</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|PCR settings|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"4\">PCR setting</th></tr><tr><th>Step</th><th>Temperature</th><th>Time</th><th>Go to</th></tr><tr><td>1</td><td>98°C</td><td>30 sec</td><td></td></tr><tr><td>2</td><td>98°C</td><td>10 sec</td><td></td></tr><tr><td>3</td><td>72°C</td><td>3 sec</td><td>step #2, 30 times</td></tr><tr><td>4</td><td>72°C</td><td>10 min</td><td></td></tr><tr><td>5</td><td>4°C</td><td>pause</td><td></td></tr></table></center></html>}}<html><br/>Since R0010 is very short, the PCR products were run on a 1.5% agarose gel using transparent loading dye.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel|<html><center><table class=\"tn-sp-table\"><tr><th>Loading scheme</th></tr><tr><td>100bp ladder</td></tr><tr><td>AHF1</td></tr><tr><td>AHF2</td></tr><tr><td>AHF3</td></tr><tr><td>AGC1</td></tr><tr><td>AGC2</td></tr><tr><td><i>empty</i></td><tr><td>1kb ladder</td></tr></tr></table><img src=\"https://static.igem.org/mediawiki/2013/3/36/Tn-20130702-GG_R0010.jpg\" alt=\"Gel\" width=\"450px\"></center></html>}}<html><br/>As shown in the gel, a band at nearly 200bp is present in each lane. So, the PCRs were successful!<br/><br/><hr><h3>(4) linear pSB1C3 amplification</h3>I didn't know that linear pSB1C3 is \"<i>impossible</i>\" to amplify, and that the only way to get it is to linearize the circular one. So I tried its amplification and failed.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"4\">Loading scheme</th></tr><tr><td>1kb ladder</td><td>G3A</td><td>G3B</td><td>G3C</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/6/6e/Tn-20130702-GG_SAMsynthetase_linearpSB1C3.jpg\" alt=\"Gel\" width=\"450px\"></center></html>}}<html></html>", | + | "content" : "<html>I performed the ligation and the trasformation of SAMsynthetase in pSB1C3 with ROO10.<br/><br/> Also I transformed Gabriele's ligations from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07- 17-gabriele- caterina\"> 17/ 07</a> into NEB10& beta;.</html>", |
| - | "tags" : "SAMsynthetase-Plac-pSB1C3" | + | "tags" : "R0010-SAMsynthetase" |
| | } | | } |
Revision as of 08:37, 3 October 2013
{
"date" : "2013-07-23",
"author" : "caterina",
"title" : "Trasformation of SAM",
"content" : "I performed the ligation and the trasformation of SAMsynthetase in pSB1C3 with ROO10.
Also I transformed Gabriele's ligations from 17/07 into NEB10β.",
"tags" : "R0010-SAMsynthetase"
}
"
