From 2013.igem.org
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| - | { | + | {"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" : "2013-07-10",
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| - | "author" : "gabriele-viola",
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| - | "title" : "Reboot: starting from the beginning",
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| - | "content" : "<html> Since the new forward primer (with the entire prefix) arrived, I started again from the beginning.<br/><br/><h3>SAM synthetase extraction</h3>The new primer forward has just the EcoRI restriction site added at its beginning (complete prefix):<br/>GAATTCGCGGCCGCTTCTAGAGAAGGAGGAACTACTATGGCAAAACACCTTTTT<br/>It has a Tm = 68.7°C.<br/><br/>Then I performed two Phusion PCR (one GC and one HF) and one Phusion/RBC PCR to identify the best protocol for SAM synthetase extraction with the new primer. </html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Phusion PCRs|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"3\">PCR Mixes</th></tr><tr><td style=\"border:none;\"></td><th>Mix HF (1)</th><th>Mix GC (2)</th></tr><tr><td>Phusion Buffer HF</td><td>10µl</td><td>0</td></tr><tr><td>Phusion Buffer GC</td><td>0</td><td>10µl</td></tr><tr><td>dNTPs</td><td colspan=\"2\">1µl</td></tr><tr><td>template</td><td colspan=\"2\">1µl</td></tr><tr><td>Primer Fw</td><td colspan=\"2\" rowspan=\"2\">2.5µl</td></tr><tr><td>Primer Rv</td></tr><tr><td>Phusion pol</td><td colspan=\"2\">0.5µl</td></tr><tr><td>Water</td><td colspan=\"2\">33.5µl</td></tr></table><br/><br/> <table class=\"tn-sp-table\"><tr><th colspan=\"4\">Phusion Settings</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>35 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></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Phusion/RBC PCR|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"2\">PCR Mix</th></tr><tr><td style=\"border:none;\"></td><td>Mix RBC (3)</td></tr><tr><td>Template</td><td>1µl</td></tr><tr><td>dNTPs</td><td>0.5µl</td></tr><tr><td>Primer Fw</td><td rowspan=\"2\">1µl</td></tr><tr><td>Primer Rv</td></tr><tr><td>Buffer RBC</td><td>5µl</td></tr><tr><td>Phusion pol</td><td>0.3µl</td></tr><tr><td>RBC</td><td>0.25µl</td></tr><tr><td>Water</td><td>40.95µl</td></tr></table><br/><br/><table class=\"tn-sp-table\"><tr><th colspan=\"4\">PCR Settings</th></tr><tr><th>Step</th><th>Temperature</th><th>Time</th><th>Go to</th></tr><tr><td>1</td><td>94°C</td><td>2 min</td><td></td></tr><tr><td>2</td><td>94°C</td><td>1 min</td><td></td></tr><tr><td>3</td><td>62.5°C</td><td>1 min</td><td></td></tr><tr><td>4</td><td>72°C</td><td>1 min 9 sec</td><td>step #2, 30 times</td></tr><tr><td>5</td><td>72°C</td><td>7 min</td><td></td></tr><tr><td>6</td><td>4°C</td><td>pause</td><td></td></tr></table></html>}}<html><br/>The products of these three PCRs were then loaded on a 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>GC(2)</td><td>HF(1)</td><td>RBC(3)</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/8/82/Tn-20130710-GG_SAMsynthetase_newPrimers_chosePCR.jpg\" alt=\"Gel\" width=\"450px\"></center></html>}}<html>As showed by the gel, only the Phusion/RBC PCR was successful.<br/><br/><hr><h3>Purifications</h3>Then, I purified the EX-SAMsynthetase-SP sample produced today with the Phusion/RBC PCR, and the 5 R0010 PCR insert that were amplified on <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07- 02-gabriele \">tuesday 02/07</a>.<center><table class=\"tn-sp- table\" ><tr><th>Sample</th><th>Type</th><th>Quantity</th></tr><tr><td>RBC(3)</td><td>EX-SAMsynthetase-SP</td><td>103.6ng/µl</td></tr><tr><td>HF1</td><td>R0010 insert</td><td>17 .5ng/µl</td></tr><tr><td>HF2</td><td>R0010 insert</td><td>19.5ng/µl</td></tr><tr><td>HF3</td><td>R0010 insert</td><td>15.9ng/µl</td></tr><tr><td>G1</td><td>R0010 insert</td><td>17.8ng/µl</td></tr><tr><td>G2</td><td>R0010 insert</td><td>16.3ng/µl</td></tr></table></center><br><hr><h3>OverNight Purification</h3>Then, Viola was so polite to prepare the O/N digestion mixes (<a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">with the usual protocol</a>) and incubate them at 37°C. An EX-SAMsynthetase-SP sample (RBC#3 from today, 103.6ng/µl) and a linear pSB1C3 sample (G3A from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-01-Gabriele\">01/07</a> , 44.8ng/& micro; l) were restricted with XbaI and PstI (Nebuffer2). </html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Digestion mixes|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>RBC#3</th><th>G3A</th></tr><tr><td>Template</td><td>40µl</td><td>50µl</td></tr><tr><td>XbaI</td><td rowspan=\"2\">2.5µl</td><td rowspan=\"2\">1.5µl</td></tr><tr><td>PstI</td></tr><tr><td>NEBuffer 2</td><td>10µl</td><td>5µl</td></tr><tr><td>BSA</td><td>10µl</td><td>5µl</td></tr><tr><td>Water</td><td>35µl</td><td>0</td></tr></table></center></html>}}<html></html>", | + | |
| - | "tags" : "SAMsynthetase"
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| - | } | + | |
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"}
"
