1208 pCDNA3.3
Cloning of 1208 pCDNA3.3
[edit]Purpose:ake pCDNA3.3 vector
Date: 2012-6-1
Notebook:
Page:
Clone ID: 1208
Experimenters: Paul
Results
[edit]cloning
[edit]6-1-2012
digest pCDNA3-YFP-IRES-SVLT 413 with Nhe1 + EcoR1 + Xba1, gel purified vector
digestion: Nhe1 + EcoR1 + Xba1
ligate to annealed pCDNA3.1-T + pCDNA3.1-B
5' CTAGC GGATCC GAATTC ggtacc AAGCTT TTAATTAA Ctcgag GCGGCCGC GATATC T 3'
3' g CCTAGG CTTAAG ccatgg TTCGAA AATTAATT Gagctc CGCCGGCG CTATAG AGATCT 5'
pCDNA3.1-T
CTAGCGGATCCGAATTCggtaccAAGCTTTTAATTAACtcgagGCGGCCGCGATATCT
pCDNA3.1-B
TCTAGAGATATCGCGGCCGCctcgaGTTAATTAAAAGCTTggtaccGAATTCGGATCCg
transform DH5alpha
6/3/2012
colony screening by miniprep: nothing
6/3/2012
Digest 1192 pCDNA3.2 (Lane 1) with Nde1 + Xba1-->insert
Digest 413 pCDNA3-YFP-IRES-SVLT (Lane 2) with Nde1 + Xba1-->vector
ligate
Colony Screening
[edit]Date: 6/4/2012
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 PacI + Nde1 to find positive clones
Gel shows samples 3-6 are positive
Digested samples 3-6 with Nhe1 + Nde1, Xba1 + Nde1 to confirm
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