Editing 1037 p8H-T7ligase (section)

== '''Cloning'''  ==

Date: 06/04/2012<br> 

PCR Template: 

[[Image:1143 1037 Template.JPG]]<br> 

Isolated band for cloning<br> 

Digested with Nde1 ad Xho1, using p8H (1135) template 

[[Image:1143 1037 Digestion.JPG]] 

Cut out bands for ligation, transformed DH5 alpha cells and grew on KAN R plates 

6/5/2012: No growth on plates, redid from digestion 

[[Image:1037 1143 Digestion 1197 1154 PCR Product.JPG]] 

Digested with&nbsp;Nde1 + Xho1, and cut of vector&nbsp;p8H (1135), purified, eluded with 20 uL water, ligated and transformed with DH5 alpha cells, grew on KAN plate over night <br> 

<br> 

06/06/2012: No growth on plates, redid PCR&nbsp;product, no product, do experiment again, maybe synthesized template incorrectly or PHUSION&nbsp;enzyme not efficient? 

[[Image:1036 Amp.JPG]] 

Redid amplication, cut out band, purified it for digestion 

[[Image:1037 amp 2.JPG]] 

Digested with Nde1 + Xho1, and cut of vector p8H (1135), purified, eluded with 20 uL water, ligated and transformed with DH5 alpha cells, grew on KAN plate over night <br> 

[[Image:06062012 Digestion.JPG]] 

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

Synthesized PCR product again, cut out circled band, purified DNA, digested DNA and vector for digestion<br> 

[[Image:1037 0607 AMP.JPG]] 

Digested with Nde1 + Xho1, and cut of vector p8H (1135), purified, eluded with 20 uL water, ligated and transformed with DH5 alpha cells, grew on KAN plate over night <br>

[[Image:0607_Digestion.JPG]]