Editing 1208 pCDNA3.3 (section)

== '''Results'''  ==

=== cloning  ===

6-1-2012<br> 

digest pCDNA3-YFP-IRES-SVLT 413 with Nhe1 + EcoR1 + Xba1, gel purified vector 

digestion: Nhe1 + EcoR1 + Xba1

<br> [[Image:1208 vector digestion 6 1 2012.jpg]]<br> 

ligate to annealed pCDNA3.1-T + pCDNA3.1-B 

5' CTAGC GGATCC GAATTC ggtacc AAGCTT TTAATTAA Ctcgag GCGGCCGC GATATC T 3'<br>3' g CCTAGG CTTAAG ccatgg TTCGAA AATTAATT Gagctc CGCCGGCG CTATAG AGATCT 5' 

pCDNA3.1-T<br>CTAGCGGATCCGAATTCggtaccAAGCTTTTAATTAACtcgagGCGGCCGCGATATCT<br>pCDNA3.1-B<br>TCTAGAGATATCGCGGCCGCctcgaGTTAATTAAAAGCTTggtaccGAATTCGGATCCg<br> 

transform DH5alpha 

6/3/2012<br> 

colony screening by miniprep: nothing<br> 

6/3/2012<br> 

Digest 1192 pCDNA3.2&nbsp; (Lane 1) with Nde1 + Xba1--&gt;insert<br> 

Digest 413 pCDNA3-YFP-IRES-SVLT (Lane 2) with Nde1 + Xba1--&gt;vector<br> 

ligate 

[[Image:1208 6 3 12012.jpg]] 

[[Image:1208 6 3 12012 1.jpg]] 

<br> 

=== Colony Screening  ===

Date: 6/4/2012<br> 

[[Image:1208 PCR Screening.JPG]] 

Positive yield for all colonies, grew lanes 1-7 overnight in AMP

Date: 6/5/2012

Did MiniPrep for samples 1-7, digested samples with&nbsp;PacI + Nde1 to find positive clones

[[Image:1208_Digestion.JPG]]

Gel shows samples 3-6 are positive

Digested samples 3-6 with&nbsp;Nhe1 + Nde1, Xba1 + Nde1 to confirm

[[Image:1208_Digestion_2.JPG]]

Lanes 2-5 were digested with Nhe1 + Nde1 and lanes 6-9 were digested with Xba1 + Nde1 (samples 3, 4, 5, 6, 3, 4, 5, 6, respectively)

Plasmids were labled, and a glycerol stock was prepared from sample 5 with AMP LB