From 2013.igem.org
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| | { | | { |
| - | "date" : "2013-06-17", | + | "date" : "2013-06-14", |
| - | "author" : "gabriele-emil", | + | "author" : "caterina", |
| - | "title" : "SAMsynthetase: ''like the legend of the phoenix,''''all ends with beginning''", | + | "title" : "Digestion& ligation of BMST1 (BBa_J45119) and PCHA (BBa_J45319)", |
| - | "content" : "<html>Today we started anew extracting SAMsynthetase from the genome of <i>E. coli</i> strain MG1655, following the usual <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#SAM-extraction-genome-coli\">protocol</a> (2 samples for Gabriele and 2 for Emil). Then we performed an electrophoresis on a 1% agarose gel.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel Result|<html><center><table class=\"tn-sp-table\"><tr><td colspan=\"5\">Loading scheme</td></tr><tr><td>G1</td><td>G2</td><td>1kb ladder</td><td>E1</td><td>E2</td></tr></table><br/><img src=\"https://static.igem.org/mediawiki/2013/a/a0/Tn-20130617-SAM_pcr_fromgenome_1706_XSP_gget.jpg\" /></center></html>}}<html><br/>Sadly, something went wrong with G2 sample (probably Gabriele forgot to add something to the PCR mix). <i>But the gel is very beautiful!!!</i><br/><br/>Then G1, E1 and E2 samples were purified using <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Promega-PCR-Gel\">Wizard® SV Gel and PCRClean-Up System</a> and then quantified using the Nanodrop.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Quantification results|<html><center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantity</th></tr><tr><td>G1</td><td>80.2ng/µl</td></tr><tr><td>E1</td><td>65.7ng/µl</td></tr><tr><td>E2</td><td>60ng/µl</td></tr></table></center></html>}}<html>Sadly, the quantities were not so high, but the results was good anyway!<br/><br/>Finally we prepared overnight digestion of SAMsynthetase (G1 and E1, E2 was put at -20°C) and pSB1C3 linearized both with XbaI and PstI-HF, using the <a href=\"\">digestion protocol</a>. We used Nebuffer4 and the mix were prepared as follows:</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Digestion mix|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border: none;\"></td><th>G1</th><th>E1</th><th>linear pSB1C3</th></tr><tr><td>Nebuffer4</td><td colspan=\"2\">10µl</td><td>5µl</td></tr><tr><td>XbaI</td><td colspan=\"3\">1µl</td></tr><tr><td>PstI-HF</td><td colspan=\"3\">1µl</td></tr><tr><td>BSA</td><td colspan=\"2\">1µl [from 100X stock]</td><td>5µl [from 10X stock]</td></tr><tr><td>DNA [3µg]</td><td>37.41µl</td><td>45.66µl</td><td>37.31µl</td></tr><tr><td>Water</td><td>49.59µl</td><td>41.34µl</td><td>0.69µl</td></tr><tr><td>Total</td><td colspan=\"2\">100µl</td><td>50µl</td></tr></table></center></html>}}<html>The mix were incubated at 37°C overnight.</html>", | + | "content" : "<html>I digested the pSB1C3 linearized with EcoR I and Spe I. <br/>Next I perfomed six different ligations:<br/>two controls with: the pSB1C3 linearized and not but without the insert<br/>two with: pSB1C3 linearized or not and BMST1<br/>two with: pSB1C3 linearized or not and PCHA</html>", |
| - | "tags" : "SAMsynthetase" | + | "tags" : "BSMT1-PchA" |
| | } | | } |
Latest revision as of 07:47, 3 October 2013
{
"date" : "2013-06-14",
"author" : "caterina",
"title" : "Digestion& ligation of BMST1 (BBa_J45119) and PCHA (BBa_J45319)",
"content" : "I digested the pSB1C3 linearized with EcoR I and Spe I.
Next I perfomed six different ligations:
two controls with: the pSB1C3 linearized and not but without the insert
two with: pSB1C3 linearized or not and BMST1
two with: pSB1C3 linearized or not and PCHA",
"tags" : "BSMT1-PchA"
}
"
