From 2013.igem.org
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| | { | | { |
| - | "date" : "2013-07-25", | + | "date" : "2013-07-04", |
| - | "author" : "gabriele", | + | "author" : "thomas", |
| - | "title" : "Screening - GOT IT! F**k yeah!", | + | "title" : "Ethylene production kinetic assay", |
| - | "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\">4.6& micro; l</td><td>3. 17µl</td></tr><tr><td>PstI-HF</td><td colspan=\"3\" rowspan=\"2\">1µl</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>", | + | "content" : "<html> In order to see the progress of the production of ethylene in time, 7 ml of culture were grown until O.D. 600 reached 0,5. The culture was then induced with 70 ul arabinose (5mM) and putted with a stirrer into a sealed tube with a pierceable septum. The tube was then connected to the Micro GC ( Agilent 3000A) and immersed in a thermal bath at 37& deg; C with magnetic stirring. A measurment was taken every hour for 8 hours. The results were not so exciting. The colony didn't produce any level ethylene and we have no idea why. Maybe the colony from which the inocula were done wasn't the right one. I'll try the experiment again the next monday! <br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler| Apparatus image|<html><center><img src=\"https://static.igem.org/mediawiki/2013/ 9/ 96/Tn- 2013_EFE_kinetic_apparatus.jpg\" style =\"width : 450px\"></center></html>}}<html></html>", |
| - | "tags" : "SAMsynthetase" | + | "tags" : "EFE" |
| | } | | } |
Revision as of 08:23, 3 October 2013
{
"date" : "2013-07-04",
"author" : "thomas",
"title" : "Ethylene production kinetic assay",
"content" : "In order to see the progress of the production of ethylene in time, 7 ml of culture were grown until O.D. 600 reached 0,5. The culture was then induced with 70 ul arabinose (5mM) and putted with a stirrer into a sealed tube with a pierceable septum. The tube was then connected to the Micro GC (Agilent 3000A) and immersed in a thermal bath at 37°C with magnetic stirring. A measurment was taken every hour for 8 hours. The results were not so exciting. The colony didn't produce any level ethylene and we have no idea why. Maybe the colony from which the inocula were done wasn't the right one. I'll try the experiment again the next monday!
",
"tags" : "EFE"
}
"
![](\"https://static.igem.org/mediawiki/2013/9/96/Tn-2013_EFE_kinetic_apparatus.jpg\")
