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| - | { | + | {"date":"2013-07- 01","author":" viola","title":" <html>PCR of the double terminator BBa_B0015</html>","content":"<html> Today i performed two PCR of the terminators both from the template in pSB1AK3 and from pSB1C£ following this quantities: <table> <tr> <th></th> <th> B0015 in pSB1AK3 < br/> 219,4 ng/ µl </ th> < th> B0015 in pSB1C3 <br/> 413, 3 ng/ µl </th></tr> <tr> <td> H20</td> <td> 33.3 µl </td> <td> 33.3 µl</td> </tr> <tr> <td> DNA</td> <td>0.5 µl</td> <td>0.5 µl</td> </tr> <tr> <td> DNTPs</td> <td> 1 µl</td> <td>1 µl</td> </tr> <tr> <td> pref. FW</td> <td>2.5 µl</td> <td>2.5 µl</td> </tr> <tr> <td> suff. RV</td> <td >2.5 µl</td> <td>2.5 µl</td> </tr> <tr> <td> BUFFER</td> <td> 10 µl</td> <td>10 µl</td> </tr> <tr> <td> Phusion pol.</td> <td> 0.5 µl</td> <td> 0.5 µl</td> </tr> </table> </html>}}< /html> \" I loaded the samples without dye and with 30% glycerol and this is the picture of the gel :</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel image|<html><center><img src=\"https://static.igem.org/mediawiki/2013/3/ 37/Tn- 2013_Corsa20minPCR_B0015.jpg\" width=\"450px\" /></center></html>}}<html> on the left of the central 100bp ladder ther is B0015 in psb1ak3 and on the right there is B0015 in psb1c3.","tags":" EFE"} |
| - | "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 1µl of DpnI to the SAMsynthetase#G2 O/N digestion and 1µl of SAP to the linear_pSB1C3#G2 digestion and then incubated them at 37°C for 1.5 hours (afterward I inhibited the enzyme with 20 minutes at 80°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/µl</td></tr><tr><td>G2 <i>linear pSB1C3 </i></td><td>6.5ng/µl</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 µl</td ></tr><tr><td> Plasmid</td><td colspan=\"3\">14.3 µl</td></tr><tr><td> Insert</td><td>0 </td><td>6µl</td><td>12µl</td></tr><tr><td>Ligase</td><td colspan=\"3\">1µl</td></tr><tr><td>Water</td><td>11. 7µl</td><td>5 .7µl</td><td>0 µl</td></tr></table></center><br/>And, \"finally\", I transformed the ligations in NEB10β 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/µl) 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°C) and the suffix Rv (Tm = 90°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>10µl</td></tr><tr><td>Phusion HF buffer</td><td>10µl</td><td>0</td></tr><tr><td>dNTPs</td><td colspan=\"2\">1 µl</td></tr><tr><td> primer Fw</td><td colspan=\"2\">2.5 µl</td ></tr><tr><td >primer Rv</td><td colspan=\"2\">2.5 µl</td></tr><tr><td> template (50ng/µl)</td><td colspan=\"2 \">0.5 µl</td ></tr><tr><td> Phusion pol</td><td colspan=\"2 \">0.5 µl</td></tr><tr><td> Water</td><td colspan=\"2\"> 33µl</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°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> 3 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> <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"
| + | |
| - | } | + | |
Latest revision as of 10:32, 3 October 2013
{"date":"2013-07-01","author":"viola","title":"PCR of the double terminator BBa_B0015","content":"Today i performed two PCR of the terminators both from the template in pSB1AK3 and from pSB1C£ following this quantities:
| B0015 in pSB1AK3
219,4 ng/ µl | B0015 in pSB1C3
413,3 ng/ µl |
| H20 | 33.3 µl | 33.3 µl |
| DNA | 0.5 µl | 0.5 µl |
| DNTPs | 1 µl | 1 µl |
| pref. FW | 2.5 µl | 2.5 µl |
| suff. RV | 2.5 µl | 2.5 µl |
| BUFFER | 10 µl | 10 µl |
| Phusion pol. | 0.5 µl | 0.5 µl |
}}</html>\" I loaded the samples without dye and with 30% glycerol and this is the picture of the gel :</html>
on the left of the central 100bp ladder ther is B0015 in psb1ak3 and on the right there is B0015 in psb1c3.","tags":"EFE"}
"
![](\"https://static.igem.org/mediawiki/2013/3/37/Tn-2013_Corsa20minPCR_B0015.jpg\")
