Team:EPF-Lausanne/Notebook/3 August 2012

From 2012.igem.org

(Difference between revisions)
(Created page with "{{:Team:EPF-Lausanne/Template/Header}} {{:Team:EPF-Lausanne/Template/NotebookHeader}} <!-- Content between here --> == Digestion of PMP and PGL4.30 for ligation == prepared P...")
Line 35: Line 35:
BSA 100x 0.5µL
BSA 100x 0.5µL
Water 41.5 µL
Water 41.5 µL
 +
 +
 +
PMP2 for ligation
 +
 +
DNA 19µL
 +
SpeI 1µL
 +
NotI 1µL
 +
N2 buffer 10x 5µL
 +
BSA 100x 0.5µL
 +
Water 23.5 µL
 +
 +
PGL2 for ligation
 +
 +
DNA 12µL
 +
HindIII 1µL
 +
MfeI 1µL
 +
N2 buffer 10x 5µL
 +
BSA 100x 0.5µL
 +
Water 31 µL
 +
 +
PGL3 for ligation
 +
 +
DNA 16µL
 +
HindIII 1µL
 +
FseI 1µL
 +
N2 buffer 10x 5µL
 +
BSA 100x 0.5µL
 +
Water 26.5 µL
All let 1:30 hours at 37ºC, shaking (600 rpm).
All let 1:30 hours at 37ºC, shaking (600 rpm).
Line 43: Line 71:
After we ran the digestion products in a gel (1% agarose, 50 µl of digestion product + 10 µl of loading dye).
After we ran the digestion products in a gel (1% agarose, 50 µl of digestion product + 10 µl of loading dye).
 +
 +
[[File:Team-EPF-Lausanne-gel03082012.jpg|thumb|Gel with digested backbones and pcr products for readouts]]
 +
<addhtml>
 +
<?xml version="1.0" encoding="UTF-8"?>
 +
<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.1 plus MathML 2.0//EN" "http://www.w3.org/Math/DTD/mathml2/xhtml-math11-f.dtd">
 +
<html xmlns="http://www.w3.org/1999/xhtml"><!--This file was converted to xhtml by OpenOffice.org - see http://xml.openoffice.org/odf2xhtml for more info.--><head profile="http://dublincore.org/documents/dcmi-terms/"><meta http-equiv="Content-Type" content="application/xhtml+xml; charset=utf-8"/><title xml:lang="en-US">- no title specified</title><meta name="DCTERMS.title" content="" xml:lang="en-US"/><meta name="DCTERMS.language" content="en-US" scheme="DCTERMS.RFC4646"/><meta name="DCTERMS.source" content="http://xml.openoffice.org/odf2xhtml"/><meta name="DCTERMS.creator" content="david "/><meta name="DCTERMS.issued" content="2012-08-06T15:33:17" scheme="DCTERMS.W3CDTF"/><meta name="DCTERMS.provenance" content="" xml:lang="en-US"/><meta name="DCTERMS.subject" content="," xml:lang="en-US"/><link rel="schema.DC" href="http://purl.org/dc/elements/1.1/" hreflang="en"/><link rel="schema.DCTERMS" href="http://purl.org/dc/terms/" hreflang="en"/><link rel="schema.DCTYPE" href="http://purl.org/dc/dcmitype/" hreflang="en"/><link rel="schema.DCAM" href="http://purl.org/dc/dcam/" hreflang="en"/><style type="text/css">
 +
@page {  }
 +
table { border-collapse:collapse; border-spacing:0; empty-cells:show }
 +
td, th { vertical-align:top; font-size:10pt;}
 +
h1, h2, h3, h4, h5, h6 { clear:both }
 +
ol, ul { margin:0; padding:0;}
 +
li { list-style: none; margin:0; padding:0;}
 +
<!-- "li span.odfLiEnd" - IE 7 issue-->
 +
li span. { clear: both; line-height:0; width:0; height:0; margin:0; padding:0; }
 +
span.footnodeNumber { padding-right:1em; }
 +
span.annotation_style_by_filter { font-size:95%; font-family:Arial; background-color:#fff000;  margin:0; border:0; padding:0;  }
 +
* { margin:0;}
 +
.ta1 { writing-mode:lr-tb; }
 +
.Default { font-family:Arial; }
 +
.co1 { width:0.889in; }
 +
.co10 { width:2.0819in; }
 +
.co2 { width:2.548in; }
 +
.co3 { width:1.802in; }
 +
.co4 { width:1.9425in; }
 +
.co5 { width:1.7402in; }
 +
.co6 { width:1.4756in; }
 +
.co7 { width:1.911in; }
 +
.co8 { width:1.4291in; }
 +
.co9 { width:1.989in; }
 +
.ro1 { height:0.1681in; }
 +
.ro2 { height:0.178in; }
 +
<!-- ODF styles with no properties representable as CSS -->
 +
{ }
 +
</style></head><body dir="ltr" style="writing-mode:lr-tb; "><table border="0" cellspacing="0" cellpadding="0" class="ta1"><colgroup><col width="99"/><col width="283"/><col width="200"/><col width="216"/><col width="193"/><col width="164"/><col width="212"/><col width="159"/><col width="221"/><col width="231"/></colgroup><tr class="ro1"><td style="text-align:left;width:0.889in; " class="Default"><p>ladder </p></td><td style="text-align:left;width:2.548in; " class="Default"> </td><td style="text-align:left;width:1.802in; " class="Default"><p>PMP2 cut w/ NotI SpeI</p></td><td style="text-align:left;width:1.9425in; " class="Default"> </td><td style="text-align:left;width:1.7402in; " class="Default"><p>PGL2 cut w/ HindII MfeI</p></td><td style="text-align:left;width:1.4756in; " class="Default"> </td><td style="text-align:left;width:1.911in; " class="Default"><p>PGL3 cut w/ HindII FseI</p></td><td style="text-align:left;width:1.4291in; " class="Default"> </td><td style="text-align:left;width:1.989in; " class="Default"><p>control of HindII w/ PGL3</p></td><td style="text-align:left;width:2.0819in; " class="Default"><p>control of FseI w/ PGL3</p></td></tr><tr class="ro2"><td style="text-align:left;width:0.889in; " class="Default"> </td><td style="text-align:left;width:2.548in; " class="Default"> </td><td style="text-align:left;width:1.802in; " class="Default"> </td><td style="text-align:left;width:1.9425in; " class="Default"> </td><td style="text-align:left;width:1.7402in; " class="Default"> </td><td style="text-align:left;width:1.4756in; " class="Default"> </td><td style="text-align:left;width:1.911in; " class="Default"> </td><td style="text-align:left;width:1.4291in; " class="Default"> </td><td style="text-align:left;width:1.989in; " class="Default"> </td><td style="text-align:left;width:2.0819in; " class="Default"> </td></tr><tr class="ro1"><td style="text-align:left;width:0.889in; " class="Default"><p>ladder </p></td><td style="text-align:left;width:2.548in; " class="Default"><p>control w/ HindIII and XbaI for PMP2</p></td><td style="text-align:left;width:1.802in; " class="Default"><p>Control of NotI w/ PMP2</p></td><td style="text-align:left;width:1.9425in; " class="Default"><p>Control of SpeI w/ PMP2</p></td><td style="text-align:left;width:1.7402in; " class="Default"> </td><td style="text-align:left;width:1.4756in; " class="Default"><p>eGFP pcr product</p></td><td style="text-align:left;width:1.911in; " class="Default"><p>TNFR pcr product</p></td><td style="text-align:left;width:1.4291in; " class="Default"><p>SEAP pcr product</p></td><td style="text-align:left;width:1.989in; " class="Default"> </td><td style="text-align:left;width:2.0819in; " class="Default"> </td></tr></table><table border="0" cellspacing="0" cellpadding="0" class="ta1"><colgroup><col width="99"/></colgroup><tr class="ro2"><td style="text-align:left;width:0.889in; " class="Default"> </td></tr></table><table border="0" cellspacing="0" cellpadding="0" class="ta1"><colgroup><col width="99"/></colgroup><tr class="ro2"><td style="text-align:left;width:0.889in; " class="Default"> </td></tr></table></body></html>
 +
</addhtml>

Revision as of 14:07, 6 August 2012



Digestion of PMP and PGL4.30 for ligation

prepared PMP2 , PGL2 and PGL3 for ligation

restriction enzyme digest mixtures were as follows:

PMP2 control for plasmid

DNA 2µL HindIII 1µL XbaI 1µL N4 buffer 10x 5µL BSA 100x 0.5µL Water 40.5 µL

PMP2 control for NotI

DNA 2µL NotI 1µL N4 buffer 10x 5µL BSA 100x 0.5µL Water 41.5 µL

PMP2 control for SpeI

DNA 2µL SpeI 1µL N4 buffer 10x 5µL BSA 100x 0.5µL Water 41.5 µL


PMP2 for ligation

DNA 19µL SpeI 1µL NotI 1µL N2 buffer 10x 5µL BSA 100x 0.5µL Water 23.5 µL

PGL2 for ligation

DNA 12µL HindIII 1µL MfeI 1µL N2 buffer 10x 5µL BSA 100x 0.5µL Water 31 µL

PGL3 for ligation

DNA 16µL HindIII 1µL FseI 1µL N2 buffer 10x 5µL BSA 100x 0.5µL Water 26.5 µL

All let 1:30 hours at 37ºC, shaking (600 rpm).


Bands got in the control of DNA and enzymes. Everything seems to be correct now!

After we ran the digestion products in a gel (1% agarose, 50 µl of digestion product + 10 µl of loading dye).


Gel with digested backbones and pcr products for readouts


<addhtml>


<?xml version="1.0" encoding="UTF-8"?> <!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.1 plus MathML 2.0//EN" "http://www.w3.org/Math/DTD/mathml2/xhtml-math11-f.dtd"> - no title specified

ladder

 

PMP2 cut w/ NotI SpeI

 

PGL2 cut w/ HindII MfeI

 

PGL3 cut w/ HindII FseI

 

control of HindII w/ PGL3

control of FseI w/ PGL3

          

ladder

control w/ HindIII and XbaI for PMP2

Control of NotI w/ PMP2

Control of SpeI w/ PMP2

 

eGFP pcr product

TNFR pcr product

SEAP pcr product

  
 
 

</addhtml>




Protocol: None

Forgot to insert protocol.


Comments

Insert comments about what happened.


Wiki Links

Important pages

Pages

Retrieved from "http://2012.igem.org/Team:EPF-Lausanne/Notebook/3_August_2012"