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Team:UNITN-Trento/Notebook/Labposts/08/18
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(Created page with "{ "date" : "2013-08-11", "author" : "thomas-viola", "title" : "Overnight cultures", "content" : "<html>Starting with the plates we did <a href=\"https://2013.igem.org/wiki/ind...") |
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{ | { | ||
| - | "date" : "2013-08- | + | "date" : "2013-08-07", |
| - | "author" : "thomas | + | "author" : "thomas", |
| - | "title" : " | + | "title" : "pXil + GFP transformation in B. subtilis", |
| - | "content" : "<html> | + | "content" : "<html>In order to transform <i>Bacillus</i> with pXyl (BBa_K823024) + GFP (BBa_E0840), reamplified and screened <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-08-02-thomas\">the 2nd of August</a>, I prepared an inoculum in 2 ml of MN completed medium (<a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#subtilis-transformation\">see protocol</a>). While I was waiting that O.D.600 reached 0.5, I proceeded digesting 2,5µg of sample 2 and sample 3 of the construct pXyl + GFP using ScaI as restriction enzyme. I did this in order to linearize the plasmid and allow his integration in <i>B. subtilis</i> genome. 0.5µg of each sample were then run on an agarose gel for screening.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel image|<html><center><img src=\"https://static.igem.org/mediawiki/2013/9/90/Tn-2013_gel_pXyl%2BGFP_ScaI_digested.JPG\" width=\"450px\" /></center></html>}}<html> As you can see from the picture, there are two not expected bands: one at height of 4500 bp and one at height of about 1800 bp. I can't explain this two bands since ScaI should digest only at only one sites on the construct.. However there's also a band at the height between 5000 and 6000 bp as expected (pXyl+GFP = 5782 bp) so I transformed it into <i>bacillus</i> anyway.</html>", |
| - | "tags" : " | + | "tags" : "pXyl-GFP-B. subtilis" |
} | } | ||
Latest revision as of 09:07, 3 October 2013
{ "date" : "2013-08-07", "author" : "thomas", "title" : "pXil + GFP transformation in B. subtilis", "content" : "In order to transform Bacillus with pXyl (BBa_K823024) + GFP (BBa_E0840), reamplified and screened the 2nd of August, I prepared an inoculum in 2 ml of MN completed medium (see protocol). While I was waiting that O.D.600 reached 0.5, I proceeded digesting 2,5µg of sample 2 and sample 3 of the construct pXyl + GFP using ScaI as restriction enzyme. I did this in order to linearize the plasmid and allow his integration in B. subtilis genome. 0.5µg of each sample were then run on an agarose gel for screening.
As you can see from the picture, there are two not expected bands: one at height of 4500 bp and one at height of about 1800 bp. I can't explain this two bands since ScaI should digest only at only one sites on the construct.. However there's also a band at the height between 5000 and 6000 bp as expected (pXyl+GFP = 5782 bp) so I transformed it into bacillus anyway.", "tags" : "pXyl-GFP-B. subtilis" }![](\"https://static.igem.org/mediawiki/2013/9/90/Tn-2013_gel_pXyl%2BGFP_ScaI_digested.JPG\")
