From 2013.igem.org
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| | { | | { |
| - | "date" : "2013-06-18", | + | "date" : "2013-06-14", |
| - | "author" : "gabriele-emil", | + | "author" : "fabio-bruno", |
| - | "title" : "SAMsynthetase: episode 2<html><br/></html>''Attack of the Insert''", | + | "title" : "bacillus subtilis promoters (part 1)!!", |
| - | "content" : "<html>This morning we added 1µl of SAP to the pSB1C3 overnight digestion and 1µl of DpN1 to the SAMsynthetase overnight digestion, then both were incubated at 37°C for 1.5 hours.<br/><br/>During these 1.5 hours, we performed the miniprep (<a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Promega-PCR-Gel\">protocol</a>) and quantification of the circular pSB1C3 inocula of yesterday.<br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Circular pSB1C3 Inocula (Gabriele)|<html><center><img src=\"https://static.igem.org/mediawiki/2013/5/5e/Tn-20130618-pSB1C3_circular_inocula_gg.JPG\" width=\"450px\" /></center></html>}}<html></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Circular pSB1C3 Inocula (Emil)|<html><center><img src=\"https://static.igem.org/mediawiki/2013/4/4a/Tn-20130618-pSB1C3_circular_inocula_et.JPG\" height=\"450px\" /></center></html>}}<html></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Circular pSB1C3 quantification results|<html><center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantity</th></tr><tr><td>G1</td><td>184.1 ng/µl</td></tr><tr><td>G2</td><td>187.7 ng/µl</td></tr><tr><td>G3</td><td>165.7 ng/µl</td></tr><tr><td>E1</td><td>117.3 ng/µl</td></tr><tr><td>E2</td><td>105.7 ng/µl</td></tr><tr><td>E3</td><td>99.0 ng/µl</td></tr></table></center></html>}}<html><br/>After the incubation, we purified the digestion mixes using the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Promega-PCR-Gel\">Wizard® SV Gel and PCR Clean-Up System</a> and then quantified.</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>linear pSB1C3</td><td>30.0 ng/µl</td></tr><tr><td>SAMsynthetase G1</td><td>40.4 ng/µl</td></tr><tr><td>SAMsynthetase E1</td><td>32.8 ng/µl</td></tr></table><br/>SAMsynthetase E1 was stocked at -20°C.</center></html>}}<html><br/>Then, we performed the ligation of pSB1C3 and SAMsynthetase exploiting the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Ligation\">usual protocol</a>.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Ligation mix|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border: none;\"></td><th>Ctrl</th><th>1:1</th><th>1:2</th><th>1:4</th></tr><tr><th>Buffer</th><td colspan=\"3\">2.5µl</td><td>3.0µl</td></tr><tr><th>Plasmid</th><td colspan=\"4\">10µl</td></tr><tr><th>Insert</th><td>0</td><td>4.14µl</td><td>8.28µl</td><td>16.56µl</td></tr><tr><th>Ligase</th><td colspan=\"4\">2µl</td></tr><tr><th>Water</th><td>10.5µl</td><td>6.36µl</td><td>2.22µl</td><td>0</td></tr><tr><th>Total</th><td colspan=\"4\">25µl</td></tr></table></center></html>}}<html>We incubated the ligation mixes for 2 hours at room temperature.<br/><br/>Also, we extracted R0010 promoter (Plac) from the registry (2013 distribution kit, plate 3, well 3H). Unfortunately, we also extracted a part from the same plate and well of the 2012 distribution kit (BBa_K115032) because we mistook the kits.<br/><br/>Finally, we transformed NEB10β competent cells with the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Competent-cells-transformation\">usual protocol</a>, we used the following quantities of DNA: 10µl of each ligation product except for the 1:4 ligation, of which we used 15µl, and 1µl of the extracted R0010. Then, we plated on CM plates.</html>", | + | "content" : "<html>First, we extracted K090504 and K090501 (gram+ consitutive promoter and gram+ IPTG inducible promoter)from 2012 kit n5. Then we tried to transform 2 ul of them in 100 ul of NEB10B cells. Furthermore we transformed 3 ul of K823000, K823002, K823003, K143012 in 200 ul of NEB10B. </html>", |
| - | "tags" : "SAMsynthetase" | + | "tags" : "Pveg-PliaG-PlepA" |
| | } | | } |
Latest revision as of 07:47, 3 October 2013
{
"date" : "2013-06-14",
"author" : "fabio-bruno",
"title" : "bacillus subtilis promoters (part 1)!!",
"content" : "First, we extracted K090504 and K090501 (gram+ consitutive promoter and gram+ IPTG inducible promoter)from 2012 kit n5. Then we tried to transform 2 ul of them in 100 ul of NEB10B cells. Furthermore we transformed 3 ul of K823000, K823002, K823003, K143012 in 200 ul of NEB10B. ",
"tags" : "Pveg-PliaG-PlepA"
}
"
