Team:UNITN-Trento/Notebook/Labposts/08/46

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{
{
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"date" : "2013-08-19",
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"date" : "2013-08-17",
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"author" : "bruno",
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"author" : "emil",
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"title" : "Target acquired, update in progress, that the mutagenesis begin",
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"title" : "To be continued 2 ",
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"content" : "I used the specific primer degnigned and created to insert RBS in the K952003 part before the amilGFP protein. For doing that I phosphorylated the primer with PNK chinase. In addition I doing 2 PCR to mutagenize the sequence using 100pg and 1ng of DNA template. The condition how the PCR works are: 67 degree of anealing, 1.30 mins of extention time and 1.5 ul of DMSO. It took the life the part K1065302",
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"content" : "<html> I verified that the transformation has succeded only in one case (1:1) with 4 colonies so I performed the miniprep following the <a href='https://2013.igem.org/Team:UNITN-Trento/Protocols#miniprep'> protocol </a>, after that I tested the construct by digestion with Sca1 following the Thermo scientific  protocol for Sca1 digestion  with the following result:</html> {{:Team:UNITN-Trento/Templates/Styles/Spoiler|gel image|<html> <img src='https://static.igem.org/mediawiki/2013/b/b8/Tn-2013_Foto_%288%29.JPG' width='400px;'></html>}}<html> as you can see in the second lane ther is a single  bright band at 6000 bp which correspond to the final construct, this construct were transformed in 400 &micro;l <i>Bacillus</i> cells growth at O.D. 1.1 in Minimal medium following the <a href='https://2013.igem.org/Team:UNITN-Trento/Protocols#subtilis-transformation'> transformation protocol </a>. These cells were plated on Spectinomicin added Agar.</html>",
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"tags" : "Blue light, mutagenesis"
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"tags" : "pXyl-GFP"
}
}

Latest revision as of 09:15, 3 October 2013

{ "date" : "2013-08-17", "author" : "emil", "title" : "To be continued 2 ", "content" : " I verified that the transformation has succeded only in one case (1:1) with 4 colonies so I performed the miniprep following the protocol , after that I tested the construct by digestion with Sca1 following the Thermo scientific protocol for Sca1 digestion with the following result:

gel image
as you can see in the second lane ther is a single bright band at 6000 bp which correspond to the final construct, this construct were transformed in 400 µl Bacillus cells growth at O.D. 1.1 in Minimal medium following the transformation protocol . These cells were plated on Spectinomicin added Agar.", "tags" : "pXyl-GFP" }

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