Editing 1155 pET28b-Ara-S1

== '''Cloning of Arabidopsis S1 Nuclease into&nbsp;1155 pET28b'''  ==

'''Purpose:''' Cloning of&nbsp;Arabidopsis S1 Nuclease into&nbsp;1155 pET28b 

'''Date:&nbsp;''' 06/05/2012<br> 

'''Notebook:&nbsp;''' 

'''Page: 008803-003308''' 

'''Clone ID: 1155''' 

'''Experimenters:'''&nbsp; Jennifer Truong<br> 

== '''Cloning'''  ==

See [http://www.excellgen.net/eln/index.php/1154_pCAMBIA-2301-Ara-s1 1154]&nbsp;(mistake of name, using a different plasmid) 

6/5/2012: 1154-5 sequencing came back mutated, restarted experiment from the beginning: 

[[Image:1155 Digestion.JPG]] 

Synthesized PCR product with PHUSION enzyme, ran PCR gel, cut out product for purifaction 

[[Image:1155 PCR Product.JPG]] 

Digested 1155 and pET28b (vector) with Nco1 + Xho1, cut out bands, ligated and transformed cells with DH5 alpha and grew on KAN plates<br> 

06/06/12: Nothing grew on the KAN&nbsp;plates 

Cut out the wrong band from the day before (see above), redid gene synthesis and cut out circled band[[Image:1155 Amp.JPG]] 

Digested purified DNA and pET28b (V12)&nbsp;vector with Nco1 +&nbsp;Xho1, cut out bands for ligation 

[[Image:1155 Digestion 06062012.JPG]] 

Redid gene synthesis, cut out band, purified for digestion 

[[Image:1155 amp 2.JPG]] 

Digested purified DNA and pET28b (V12) vector with Nco1 + Xho1, cut out bands for ligation <br> 

[[Image:06062012 Digestion.JPG]] 

06/07/2012: Again, no growth on plates. 

Synthesized PCR&nbsp;product again, cut out circled band, purified DNA, digested DNA and vector for digestion 

[[Image:1155 0607 AMP.JPG]] 

<br>

Digested purified DNA and pET28b (V12) vector with Nco1 + Xho1, cut out bands for ligation (lanes 1 and 2)<br>

[[Image:0607_Digestion.JPG]]

== '''Colony Screening'''<br>  ==

Date:&nbsp;06/08/2012<br> 

[[Image:0608 PCR Screening.JPG]] 

All screening were positive, grew samples 1-6 in media (lanes 1-6) 

[[Image:1197_1155_Digestion.JPG]]

Performed miniprep on samples, digested with enzymes to check for inserts, submitted sample #2 (lane 8)&nbsp;for sequencing.

== '''Result intepretation/Summary'''  ==

<br> 

== '''Conclusion'''  ==

<br> 

[[Category:Cloning]]