Team:UNITN-Trento/Notebook/Labposts/07/28

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{"date" : "2013-07-11","author" : "gabriele","title" : "Extraction (again) and ligation","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 1&micro;l of SAP to pSB1C3 O/N digestion and 1&micro;l of DpnI to SAMsynthetase O/N digestion, and incubated the samples at 37&deg;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/&micro;l</td></tr><tr><td>pSB1C3</td><th>14.0ng/&micro;l</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\">4&micro;l</td></tr><tr><td>Plasmid</td><td colspan=\"3\">14.29&micro;l</td></tr><tr><td>Insert</td><td>0</td><td>9.15&micro;l</td><td>18.3&micro;l</td></tr><tr><td>Ligase</td><td colspan=\"3\">1&micro;l</td></tr><tr><td>Water</td><td>20.71&micro;l</td><td>11.56&micro;l</td><td>2.41&micro;l</td></tr></table></center></html>}}<html>Then I transformed the ligation products in NEB10&beta;</html>","tags" : "SAMsynthetase"}
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"date" : "2013-07-15",
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"author" : "thomas",
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"title" : "Ethylene production kinetic assay (third and last trial)",
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"content" : "<html>Since I was not satisfied with the curve obtained during the <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-08-thomas\">last experiment</a>, I decided to redo the experiment, this time starting from a culture with O.D.600 = 0,8 and registering a measurment every 45 min. <br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Plot|<html><center><img src=\"https://static.igem.org/mediawiki/2013/0/00/Tn-2013_kinetic_EFE_plot-2.png\" style =\"width: 450px\"></center></html>}}<html>Interestingly, inducing with arabinose when the sample has reached OD600 0.8 causes an increase of more than two fold in the production of ethylene.</html>",
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"tags" : "EFE"
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}
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Latest revision as of 10:38, 3 October 2013

{"date" : "2013-07-11","author" : "gabriele","title" : "Extraction (again) and ligation","content" : "

SAM synthetase extraction

Last time I determined the correct protocol to extract SAM synthetase from the E. coli using the new plasmid (at the end, the protocol was the usual one). I performed three PCRs (G1, G2, G3) and run the products on 1% agarose gel.
Gel
Loading scheme
1kb ladderG1G2G3
\"Gel\"


Ligation

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.
SampleQuantity
SAM synthetase12.2ng/µl
pSB1C314.0ng/µl

Then I performed the ligation and left the reaction run for 2h at room temperature.
Ligation Mixes
CTRL1:11:2
Buffer4µl
Plasmid14.29µl
Insert09.15µl18.3µl
Ligase1µl
Water20.71µl11.56µl2.41µl
Then I transformed the ligation products in NEB10β","tags" : "SAMsynthetase"}