From 2013.igem.org
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| | { | | { |
| - | "date" : "2013-07-11", | + | "date" : "2013-07-27", |
| - | "author" : "gabriele", | + | "author" : "thomas", |
| - | "title" : "Extraction (again) and ligation", | + | "title" : "AraCpBAD + EFE + Venus transformation", |
| - | "content" : "<html> <h3>SAM synthetase extraction</h3><a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-10-gabriele-viola\">Last time</a> I determined the correct protocol to extract SAM synthetase from the <i>E. coli</i> using the new plasmid (at the end, the protocol was <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#SAM-extraction-genome-coli\">the usual one</a>). I performed three PCRs (G1, G2, G3) and run the products on 1% agarose gel.</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>G1</td><td>G2</td><td>G3</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/ f/ f4/Tn- 20130711-GG_SAMsynthetase_extraction_EXSP. jpg\" alt= \"Gel\" title=\"Gel\" width =\"450px\" /></center></html>}}<html> <br><hr><h3>Ligation</h3>At first, I added 1µl of SAP to pSB1C3 O/N digestion and 1µl of DpnI to SAMsynthetase O/N digestion, and incubated the samples at 37°C for 1. 5h. Then I quantified the digestions.<center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantity</th></tr><tr><td>SAM synthetase</td><td>12.2ng/µl</td></tr><tr><td>pSB1C3</td><th>14.0ng/µl</th></tr></table></center><br/>Then I performed the ligation and left the reaction run for 2h at room temperature. </html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Ligation Mixes|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>CTRL</th><th>1:1</th><th>1:2</th></tr><tr><td>Buffer</td><td colspan=\"3\">4µl</td></tr><tr><td>Plasmid</td><td colspan=\"3\">14.29µl</td></tr><tr><td>Insert</td><td>0</td><td>9.15µl</td><td>18.3µl</td></tr><tr><td>Ligase</td><td colspan=\"3\">1µl</td></tr><tr><td>Water</td><td>20.71µl</td><td>11.56µl</td><td>2.41µl</td></tr></table></center></html>}}<html>Then I transformed the ligation products in NEB10β</html>", | + | "content" : "<html> Once I confirmed the construct through an electrophoresis analisis, I decided to transorm the sample C into NEB10b cells. I plated then 100 ul of cells and putted the plate in static incubation at 37°C overnight.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler| Plate Image|<html><center><img src=\"https://static.igem.org/mediawiki/2013/ 5/ 53/Tn- 2013_efe%2Bvenus_plate. JPG\" style =\"width : 450px\"></center></html>}}<html> As you can see from the picture, the colonies grown. One of them was then picked up and inoculated overnight.</html>", |
| - | "tags" : "SAMsynthetase" | + | "tags" : "EFE-Venus" |
| | } | | } |
Revision as of 08:44, 3 October 2013
{
"date" : "2013-07-27",
"author" : "thomas",
"title" : "AraCpBAD + EFE + Venus transformation",
"content" : "Once I confirmed the construct through an electrophoresis analisis, I decided to transorm the sample C into NEB10b cells. I plated then 100 ul of cells and putted the plate in static incubation at 37°C overnight.
As you can see from the picture, the colonies grown. One of them was then picked up and inoculated overnight.",
"tags" : "EFE-Venus"
}
"
![](\"https://static.igem.org/mediawiki/2013/5/53/Tn-2013_efe%2Bvenus_plate.JPG\")
