Team:UNITN-Trento/Notebook/Labposts/08/10

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(Created page with "{ "date" : "2013-08-07", "author" : "viola", "title" : "cloning cloning cloning!", "content" : "<html>The plates that i did <a href=\"https://2013.igem.org/Team:UNITN-Trento/N...")
 
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{
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"date" : "2013-08-07",
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"date" : "2013-08-05",
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"author" : "viola",
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"author" : "thomas",
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"title" : "cloning cloning cloning!",
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"title" : "EFE PCR",
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"content" : "<html>The plates that i did <a href=\"https://2013.igem.org/Team:UNITN-Trento/Notebook?title=Team:UNITN-Trento/Notebook&action=edit&section=138\">yesterday</a> are quite perfect! The ctrl plate is quite empty and there are many colonies on the other samples (1:1,1:2,1:3,1:4). This evening i will do some inocula. Today I'm making also the screening of yesterday's inocula from that bad plates and from the gel we can see that the ligation worked only in the third sample:</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel image|<html><center><img src=\"https://static.igem.org/mediawiki/2013/f/f6/GEL1.png\" width=\"450px\" /></center></html>}}<html> </html>",
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"content" : "<html>Today I performed a PCR on EFE in pUC57 vector in order to linearize it and eliminate the vector. To do this I used primers BBa_Fwd and BBa_Rev. The reaction was assembled as follow exploting both OneTaq and Phusion polymerase.<center><table><tr><th>5x One Taq Buffer</th><td>10&micro;l</td></tr><tr><th>Fwd Primer</th><td>1&micro;l</td></tr><tr><th>Rev Primer</th><td>1&micro;l</td></tr><tr><th>10 mM dNTP's</th><td>1&micro;l</td></tr><tr><th>One Taq</th><td>0.25&micro;l</td></tr><tr><th>Phusion</th><td>0.3&micro;l</td></tr><tr><th>Template DNA</th><td>0.7&micro;l (about 500 ng)</td><tr><th>H2O</th><td>35.75&micro;l</td></tr></tr></table></center>When the reaction finished, I performed an electrophoresis analysis in order to confirm the product. Kapa universal ladder was adopted.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel image|<html><center><img src=\"https://static.igem.org/mediawiki/2013/0/0f/Tn-2013_EFE-PCR_GEL.jpg\" width=\"450px\" /></center></html>}}<html> As you can see, the Gel image is quite confusing. First of all, the marker should be diffused in the sample lane (lane 1) as we can see the same band of the ladder but with a more tenuous coloration. Secondly, the thickest band was at about 1200 bp height even if EFE is 1078 bp long. I think the product has to be confirmed again...!</html>",
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"tags" : "s04617-EFE"
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"tags" : "EFE"
}
}

Latest revision as of 09:03, 3 October 2013

{ "date" : "2013-08-05", "author" : "thomas", "title" : "EFE PCR", "content" : "Today I performed a PCR on EFE in pUC57 vector in order to linearize it and eliminate the vector. To do this I used primers BBa_Fwd and BBa_Rev. The reaction was assembled as follow exploting both OneTaq and Phusion polymerase.

5x One Taq Buffer10µl
Fwd Primer1µl
Rev Primer1µl
10 mM dNTP's1µl
One Taq0.25µl
Phusion0.3µl
Template DNA0.7µl (about 500 ng)
H2O35.75µl
When the reaction finished, I performed an electrophoresis analysis in order to confirm the product. Kapa universal ladder was adopted.
Gel image
As you can see, the Gel image is quite confusing. First of all, the marker should be diffused in the sample lane (lane 1) as we can see the same band of the ladder but with a more tenuous coloration. Secondly, the thickest band was at about 1200 bp height even if EFE is 1078 bp long. I think the product has to be confirmed again...!", "tags" : "EFE" }

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