Team:UNITN-Trento/Notebook/Labposts/06/46

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Latest revision as of 07:45, 9 September 2013

{ "date" : "2013-06-21", "author" : "emil", "title" : "Linearization of pSB1C3 (Failed), purification and screening of SAMsynthethase (ligation 1:1) ", "content" : "Of the 8 inocula (pSB1C3+SAMsynthetase), only 7 succeeded and were purified with the Promega kit, following the miniprep protocol with vacuum. The quantification of the samples (one (D) showed a very strange absorbtion pattern, maybe due to contamination, so here is not presented) gave the following results:

Quantification results

Sample	Quantities
A	198 ng/µl
B	187.6 ng/µl
C	138 ng/µl
E	179.9 ng/µl
F	200.2 ng/µl
G	197.2 ng/µl

After that, I have digested 800ng of each sample with EcoR1-HF and Pst1-HF and one (F) also with Xba1 and Pst1-HF following the digestion protocol after 30min at 37°C I have run the final pruduct on a gel with no results again:

Gel Image

I have re-re-tried to linearize pSB1C3 (in triplicates) with no results (in the second line we can see some aspecific bands):

Linearization attempt 3

Exploiting the iGem 2012 kit I have transformed 2µl of the pLac promoter (R0010 in pSB1A2) in NEB10β competent cells following the transformation protocol. Afterwards, I plated them on an ampicillin-containing plate.", "tags" : "SAMsynthetase-Plac" }