Editing 2321 pFastBacDual-VSVG-CAG-Flp (section)

== '''Results'''  ==

=== CLONING  ===

PCR CAG-pH-T7 promoters from 298 pTriEx-HTNC<br>2364-CAG-Xho1-F atagcactcgagTATATGGAGTTCCGCGTTAC<br>2364-CAG-BamH1-R tgtcatggatccTATATCTCCTTTGATTGTAAATAAAATG 

<br> 

[[Image:2321 2374PCR.JPG]]<br> 

Digest with Xho1 + BamH1. 

Digest vector with Xho1 + BamH1. 

[[Image:DIGEST FOR 2321 2374.JPG]]<br> 

=== Colony Screening  ===

Date: 2014_9<br> 

Make miniprep, use&nbsp;Xho1 + BamH1 to find positive clone<br> 

[[Image:2321 2374digest.JPG]]<br> 

digest with NdeI+XhoI should get four bands,digest with XbaI should get two bands 

[[Image:2321DIGEST.JPG]] &nbsp; &nbsp;[[Image:2321DIGEST21.JPG]] &nbsp; &nbsp;[[Image:2321DIGEST3.JPG]] 

send 2321-2 and 2321-3 to sequence with '''NLS-HindIII-R'''(NLS-HindIII-R accttcctcttcttcttcaagcttacatac), poor quality or no priming<br> 

use primers to do pcr screening<br> 

2364-CAG-Xho1-F&nbsp;&nbsp; atagcactcgagTATATGGAGTTCCGCGTTAC<br> 

hIgG-Lc-BamH1-Nhe1-B&nbsp; ctagcggccatggccggctgggccgcgagtaataacaatccagcggctgccgtaggcaataggtatttcatggtggcg<br> 

[[Image:2321pcr screening.JPG]]<br>