Team:UNITN-Trento/Notebook/Labposts/07/49

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Latest revision as of 10:43, 3 October 2013

{"date" : "2013-07-22","author" : "fabio","title" : " blue and red light sensors!!","content" : " let’s get ready to screen!! This time I amplified my parts by pcr them out with OneTaq polymerase!! I started from the blue promoter (number A from the firt set). Pcr sample composition:
5X Onetaq standard reaction buffer 10 ul
10 mM dNTPs 1 ul
Forward primer 1ul
Reverse primer 1 ul
One Taq 0.25
Phusion 0.3
Template 0.5
Water up to 50 ul
Pcr program values:
Initial digestion: 94° 2min
Digestion: 94 30 sec
Annealing: 60 1 min
Annealing: 68° 15 sec
Final extention: 68° 5 min
(30 cycles from step 3 to step 5)
The screening was successful :

gel

Finally I purified the PCR and abtained a 209,8 ng/ul yieald.Meanwhile I started thinking about the ligation of the two blue parts: the idea is to use k592016 as plasmid and k592006 as an insert to put downstream . So I had to digest o/n 3 ug of 016(from the first set) with Spel and Pstl, and 006( the purified Pcr) with Xbal and Pstl. At the same time I digested R0010 with Ecorl and Spel, cause I’ll need to put it on top of everything once I have my biobrick.","tags" : " YF1_FixJ - FixK2- ho1_PcyA_cph8-OmpR"}

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+{"date" : "2013-07-22","author" : "fabio","title" : " blue and red light sensors!!","content" : "<html> let’s get ready to screen!! This time I amplified my parts by pcr them out with OneTaq polymerase!! I started from the blue promoter (number A from the firt  set). Pcr sample composition: <br>5X Onetaq standard reaction buffer  10 ul <br>10 mM dNTPs   1 ul<br>Forward primer 1ul<br>Reverse primer 1 ul<br>One Taq 0.25<br>Phusion 0.3<br>Template 0.5<br>Water up to 50 ul<br>Pcr program values: <br>Initial digestion: 94° 2min <br>Digestion: 94 30 sec<br>Annealing: 60 1 min<br>Annealing: 68° 15 sec<br>Final extention: 68° 5 min<br>(30 cycles from step 3 to step 5) <br>The screening was successful : <br> </html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|gel|<html><center><img src=\"https://static.igem.org/mediawiki/2013/0/06/Tn-2013_C.jpg\" width=\"450px\" /></center></html>}}<html>Finally I purified the PCR and abtained a 209,8 ng/ul yieald.Meanwhile I started thinking about the ligation of the two blue parts: the idea is to use k592016 as plasmid and k592006 as an insert to put downstream . So I had to digest o/n  3 ug of 016(from the first set)  with Spel and Pstl, and 006( the purified Pcr)  with Xbal and Pstl. At the same time I digested R0010 with Ecorl and Spel, cause I’ll need to put it on top of everything once I have my biobrick.</html>","tags" : " YF1_FixJ - FixK2- ho1_PcyA_cph8-OmpR"}
-	"date" : "2013-07-02",
-	"author" : "gabriele",
-	"title" : "Another busy day",
-	"content" : "<html><h3>SAM synthetase ligation in (linear) pSB1C3</h3>First of all, early in the morning I added 1&micro;l of DpnI to the SAMsynthetase#G2 O/N digestion and 1&micro;l of SAP to the linear_pSB1C3#G2 digestion and then incubated them at 37&deg;C for 1.5 hours (afterward I inhibited the enzyme with 20 minutes at 80&deg;C).<br/><br/>Then, I purified and quantified the two digestion samples:<center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantity</th></tr><tr><td>G1 <i>SAMsynthetase</i></td><td>18.8ng/&micro;l</td></tr><tr><td>G2 <i>linear pSB1C3</i></td><td>6.5ng/&micro;l</td></tr></table></center><br/>After that, I performed the ligation (the plasmid had a too low concentration).<center><table class=\"tn-sp-table\"><tr><td style=\"border:none\"></td><th>Ctrl</th><th>0.5:1</th><th>0.5:2</th></tr><tr><td>Buffer</td><td colspan=\"3\">3&micro;l</td></tr><tr><td>Plasmid</td><td colspan=\"3\">14.3&micro;l</td></tr><tr><td>Insert</td><td>0</td><td>6&micro;l</td><td>12&micro;l</td></tr><tr><td>Ligase</td><td colspan=\"3\">1&micro;l</td></tr><tr><td>Water</td><td>11.7&micro;l</td><td>5.7&micro;l</td><td>0&micro;l</td></tr></table></center><br/>And, \"finally\", I transformed the ligations in NEB10&beta; cells.<br/><br/><hr><h3>(2) SAMsynthetase amplification</h3>I amplified SAMsynthetase from the E2 sample (that was purified <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-01-Gabriele\">yesterday</a>) following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#SAM-extraction-genome-coli\">usual protocol</a>. The PCR was performed in triplicates.<br/><br/>The PCR products were run on a 1% agarose gel.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"5\">Loading scheme</th></tr><tr><td>E1A</td><td>E1B</td><td>E1C</td><td><i>empty</i></td><td>1kb ladder</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/6/6e/Tn-20130702-GG_SAMsynthetase_linearpSB1C3.jpg\" alt=\"Gel\" width=\"450px\"></center></html>}}<html><br/>As shown in the gel, a band at nearly 1200bp is present, confirming the success of the PCR.<br/><br/><hr><h3>(3) R0010 amplification</h3>I also amplified R0010 from <b>A</b> sample (R0010, 243.7ng/&micro;l) from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-06-27-gabriele-emil\">27/06</a>. Being the first time amplifying this sequence, I performed (in triplicates or duplicates) a Phusion PCR using as primers the prefix Fw (Tm = 86&deg;C) and the suffix Rv (Tm = 90&deg;C).<br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|PCR Mixes|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>Mix HF (x3)</th><th>Mix GC (x2)</th></tr><tr><td>Phusion GC buffer</td><td>0</td><td>10&micro;l</td></tr><tr><td>Phusion HF buffer</td><td>10&micro;l</td><td>0</td></tr><tr><td>dNTPs</td><td colspan=\"2\">1&micro;l</td></tr><tr><td>primer Fw</td><td colspan=\"2\">2.5&micro;l</td></tr><tr><td>primer Rv</td><td colspan=\"2\">2.5&micro;l</td></tr><tr><td>template (50ng/&micro;l)</td><td colspan=\"2\">0.5&micro;l</td></tr><tr><td>Phusion pol</td><td colspan=\"2\">0.5&micro;l</td></tr><tr><td>Water</td><td colspan=\"2\">33&micro;l</td></tr></table></center></html>}}<html><br/>Given that R0010 is 200bp long, the PCR program was the following:</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|PCR settings|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"4\">PCR setting</th></tr><tr><th>Step</th><th>Temperature</th><th>Time</th><th>Go to</th></tr><tr><td>1</td><td>98&deg;C</td><td>30 sec</td><td></td></tr><tr><td>2</td><td>98&deg;C</td><td>10 sec</td><td></td></tr><tr><td>3</td><td>72&deg;C</td><td>3 sec</td><td>step #2, 30 times</td></tr><tr><td>4</td><td>72&deg;C</td><td>10 min</td><td></td></tr><tr><td>5</td><td>4&deg;C</td><td>pause</td><td></td></tr></table></center></html>}}<html><br/>Since R0010 is very short, the PCR products were run on a 1.5% agarose gel using transparent loading dye.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel|<html><center><table class=\"tn-sp-table\"><tr><th>Loading scheme</th></tr><tr><td>100bp ladder</td></tr><tr><td>AHF1</td></tr><tr><td>AHF2</td></tr><tr><td>AHF3</td></tr><tr><td>AGC1</td></tr><tr><td>AGC2</td></tr><tr><td><i>empty</i></td><tr><td>1kb ladder</td></tr></tr></table><img src=\"https://static.igem.org/mediawiki/2013/3/36/Tn-20130702-GG_R0010.jpg\" alt=\"Gel\" width=\"450px\"></center></html>}}<html><br/>As shown in the gel, a band at nearly 200bp is present in each lane. So, the PCRs were successful!<br/><br/><hr><h3>(4) linear pSB1C3 amplification</h3>I didn't know that linear pSB1C3 is \"<i>impossible</i>\" to amplify, and that the only way to get it is to linearize the circular one. So I tried its amplification and failed.</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>G3A</td><td>G3B</td><td>G3C</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/6/6e/Tn-20130702-GG_SAMsynthetase_linearpSB1C3.jpg\" alt=\"Gel\" width=\"450px\"></center></html>}}<html></html>",
-	"tags" : "SAMsynthetase-Plac-pSB1C3"
-}