From 2013.igem.org
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| | { | | { |
| - | "date" : "2013-07-01", | + | "date" : "2013-07-23", |
| - | "author" : "Gabriele", | + | "author" : "emil", |
| - | "title" : "Too many things in one single day", | + | "title" : "The incompetent Bacillus subtilis saga: The LB transformation and amplification of pSpac +GFP", |
| - | "content" : "<html ><h3>(1) PCR: SAM synthetase amplification</h3>I amplified SAM synthetase by performing a PCR on the samples <b>G1</b> and <b>E2</b> from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-06-17-gabriele-emil\">17/06</a>.<center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantification</th></tr><tr><td>G1</td><td>80ng/µl</td></tr><tr><td>E2</td><td>60ng/µl</td></tr></table></center><br/>The PCR was performed following the usual <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#SAM-extraction-genome-coli\">SAM extraction protocol</a>.<br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|PCR mixes|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>G1 mix</th><th>E2 mix</th></tr><tr><td>Template(50ng)</td><td>0.63µl</td><td>0.83µl</td></tr><tr><td>dNTPs</td><td colspan=\"2\">0.5µl</td></tr><tr><td>primer Fw</td><td colspan=\"2\">1µl</td></tr><tr><td>primer Rv</td><td colspan=\"2\">1µl</td></tr><tr><td>buffer RBC</td><td colspan=\"2\">5µl</td></tr><tr><td>Phusion pol</td><td colspan=\"2\">0.3 µl</td></tr><tr><td>RBC pol</td><td colspan=\"2\">0.25µl</td></tr><tr><td>water</td><td>41.32µl</td><td>41.12µl</td></tr></table></center></html>}}<html><br/>PCR results were then run on a 1% agarose gel:<br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"3\">Loading scheme</th></tr><tr><td>1kb ladder</td><td>G1</td><td>E2</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/8/81/Tn-20130701-GGET_GFPpcr_SAMampli.jpg\" alt=\"Gel\" width=\"450px\"><br/></center></html>}}<html><br/>Since the gel shows two bands at nearly 1200bp, the PCR results are confirmed. The PCR products were then purified with the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Promega-PCR-Gel\">Promega kit</a>.<center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Type</th><th>Quantity</th></tr><tr><td>G1</td><td>SAM synthetase</td><td>35.6ng/µl</td></tr><tr><td>E2</td><td>SAM synthetase</td><td>31.5ng/µl</td></tr></table></center><br/><hr><h3>(2) pSB1A2+ R0010+ SAMsynthetase ligation screening</h3>Then I screened the inocula from the <a href=\"https://2013. igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-06-26-emil-gabriele\">26/06</a> ligation. First I miniprepped the inocula:<br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Miniprep quantities|<html><center><table class=\"tn-sp-table\"><tr><td rowspan=\"6\">1:1</td><td>A</td><td>490.9ng/µl </td></tr><tr><td>B</td><td>252.1ng/µl</td></tr><tr><td>C</td><td>345.6ng/µl</td></tr><tr><td>D</td><td>295.3ng/µl</td></tr><tr><td>E</td><td>315.6ng/µl</td></tr><tr><td>F</td><td>226.2ng/µl</td></tr><tr><td rowspan=\"3\">1 :3</td><td>A</td><td>345.7ng/µl</td></tr><tr><td>B</td><td>147.0ng/µl</td></tr><tr><td>C</td><td>268.0ng/µl</td></tr></table></center></html>}}<html><br/>The samples were then digested using the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">screening digestion protocol</a> and then run on a 1% agarose gel. <br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel|<html><center><table class=\"tn-sp-table\"><tr><th>Loading scheme</th></tr><tr><td>1kb ladder</td></tr><tr><td>1:1 A</td></tr><tr><td>1:1 B</td></tr><tr><td>1:1 C</td></tr><tr><td>1:1 D</td></tr><tr><td>1:1 E</td></tr><tr><td>1:1 F</td></tr><tr><td>1kb ladder</td></tr><tr><td>1:3 A</td></tr><tr><td>1:3 B</td></tr><tr><td>1:3 C</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/e/ef/Tn-20130701-GGET_GFP_PSB1A2R0010SAMsynth.jpg\" alt=\"gel\" width=\"450px\" /></center></html>}}<html><br/>Given that the gel shows only two bands (one at nearly 2kbp and the other at 200bp), SAM synthetase is not present and the ligation failed.<br/><br/><hr><h3>(3) Linear pSB1C3 purification</h3>I also purified with <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Promega-PCR-Gel\">the usual protocol</a> the linearized pSB1C3 from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-06-19-gabriele-emil-bruno\">19/06</a>.<br/><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>Sample</th><th>Type</th><th>Quantity</th></tr><tr><td>G2</td><td>linear pSB1C3</td><td>74.4ng/& micro; l</td></tr><tr><td>G3</td><td>linear pSB1C3</td><td>63.3ng/µl</td></tr><tr><td>G2A</td><td>linear pSB1C3</td><td>44.0ng/µl</td></tr><tr><td>G3A</td><td>linear pSB1C3</td><td>44.8ng/µl</td></tr><tr><td>G4A</td><td>linear pSB1C3</td><td>41.9ng/µl</td></tr></table></center><br/><hr><h3>(4) O/N Digestion</h3>Finally, I prepared an overnight digestion of the samples <b>G1</b> (<i>SAM synthetase</i>) and <b>G2</b> (<i>linear pSB1C3</i>) to try again the ligation tomorrow. I followed the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols# Digestion\"> usual protocol</a>. </html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Digestion mix|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>G1 SAMsynthetase</th><th>G2 linear pSB1C3</th></tr><tr><td>template (3µg)</td><td>48.5µl</td><td>40µl</td></tr><tr><td>XbaI</td><td>2.5µl</td><td>1.5µl</td></tr><tr><td>PstI</td><td>2.5µl</td><td>1.5µl</td></tr><tr><td>NEBuffer 2</td><td>10µl</td><td>5µl</td></tr><tr><td>water</td><td>26.5µl</td><td>0</td></tr></table></center></html>}}<html></html>", | + | "content" : "<html>I have transformed 3 groups of cells with 500 ng of pSpac+ pLac+ RFP,I grew Bacillus in LB and when it reached OD of 1. 1 I added the DNA to 400µl of cells,I let grow for 1 hour and then I plated the cell on Kanamicin plates. Afterwards I transformed Neb10& beta; with 100 ng of K823026+E0840 following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols# Competent-cells-transformation\"> transformation protocol</a>.</html>", |
| - | "tags" : "SAMsynthetase-Plac-pSB1A2-pSB1C3" | + | "tags" : "Bacillus subtilis-pSpac+GFP" |
| | } | | } |
Revision as of 08:37, 3 October 2013
{
"date" : "2013-07-23",
"author" : "emil",
"title" : "The incompetent Bacillus subtilis saga: The LB transformation and amplification of pSpac +GFP",
"content" : "I have transformed 3 groups of cells with 500 ng of pSpac+pLac+RFP,I grew Bacillus in LB and when it reached OD of 1.1 I added the DNA to 400µl of cells,I let grow for 1 hour and then I plated the cell on Kanamicin plates.Afterwards I transformed Neb10β with 100 ng of K823026+E0840 following the transformation protocol.",
"tags" : "Bacillus subtilis-pSpac+GFP"
}
"
