From 2013.igem.org
(Difference between revisions)
|
|
| Line 1: |
Line 1: |
| - | { | + | {"date" : "2013-07- 23","author" : " caterina","title" : " Trasformation of SAM","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" : " R0010-SAMsynthetase"} |
| - | "date" : "2013-07-25",
| + | |
| - | "author" : "gabriele",
| + | |
| - | "title" : "Screening - GOT IT! F**k yeah!",
| + | |
| - | "content" : "<html>I checked <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07- 24-gabriele \">yesterday</a>'s inocula and, surprisingly and sadly, 4 out of 7 were red!!! I must admit that I don't get how it is possible to have red inocula when I treated the linearized- by-PCR pSB1C3 with the DpnI enzyme... that's crazy... anyway the quantification results are the following:<br/><center><table class=\"tn-sp-table\"> <tr><th>Inocula</th><th>Quantity</th></tr><tr><td>ON 1:1 A</td><td style=\"font-color:red\">RED</td></tr><tr><td>ON 1:1 B</td><td style=\"font-color:red\">RED</td></tr><tr><td>ON 1:1 C</td><td>216.4ng/µl</td></tr><tr><td>ON 1:1 D</td><td>215.5ng/µl</td></tr><tr><td>ON 1:1 E</td><td style=\"font-color:red\">RED</td></tr><tr><td>Short 1:1</td><td style=\"font-color:red\">RED</td></tr><tr><td>Short 1:3</td><td>315.5ng/µl</td></tr></table></center><br><h3>Screening digestion</h3>Screening was performed with BamHI-HF and PstI-HF. While PstI-HF cuts at the suffix (the end of the insert), BamHI-HF is able to cut only SAMsynthetase at 102th base position. So, the screening digestion will show one band at nearly 3100bp if pSB1C3 contains Plac+RFP, at nearly 2000bp if an '<i>empty</i>' pSB1C3 is present and two bands (one at nearly 2200bp and the other at nearly 1000bp) if we have pSB1C3 with SAMsynthetase.<br></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Digestion mixes|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>11C</th><th>11D</th><th>13S</th></tr><tr><td>template</td><td colspan=\"2\">4.6µl</td><td>3.17 µl</ td></ tr><tr><td>PstI-HF</td><td colspan=\"3\" rowspan=\"2\"> 1& micro; l</td></tr><tr><td>BamHI-HF</td></tr><tr><td>NEBuffer 4</td><td colspan=\"3\" rowspan=\"2\">2µl</td></tr><tr><td>BSA</td></tr><tr><td>Water</td><td colspan=\"2\">9. 4µl</td><td>10.83µl</td></tr></table></center></html> }}<html>The screening digestions were incubated at 37°C for 1 hour and the run on a 1% agarose gel.<br/></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>11C</td><td>11D</td><td>13S</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/4/4c/Tn-20130725-GG_SAMsynthetase_GOTIT.jpg\" alt=\"Gel\" title=\"Gel\" width=\"450px\" /></center></html>}}<html>As you can see, the gel shows a band at nearly 2kbp and one at nearly 1kbp at the 13S lane, so that sample should contain the desired construct (pSB1C3+SAMsynthetase).</html>", | + | |
| - | "tags" : "SAMsynthetase"
| + | |
| - | } | + | |
Latest revision as of 10:45, 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"}
"
