373-pAd5-CMV-Dest-Flag
The primer-Rs of 373 and 373' are different.
1. Cloning of pAd5-CMV-Dest-Flag (373)
Purpose: to clone pAd5-CMV-Dest-Flag by PCR
Date: 2011-11-28 to 2011-12-
Notebook: Xiaojing Wang
Page: 004127---004131
Clone ID: 373
Experimenters: Xiaojing Wang
Results
The right bands show PCR product of 373,
Primer:
ad-GW-F
Kpn1
aactc GGTACC ATCAACAAGTTTGTACAAAAAAG
ad-GW-Flag-R
Xho1
(TCTTGTACAAAGTGGTTGATGATTACAAGGATGACGACGATAAGtgaCTCGAGatcga)
(tcttgtacaaagtggttgatgattacaaggatgacgacgataagtgactcgagatcga)
( L Y K V V D D Y K D D D D K - )
tcgatCTCGAGtcaCTTATCGTCGTCATCCTTGTAATCATCAACCACTTTGTACAAGA
Digest result:
digest PCR product and vector with Kpn I+Xho I,run gel
the left picture shows 373 insert, the right picture shows 373 vector (pAd5-CMV-MCS, 280)
gel purify, ligate, transform ccdb survival,AMP
Colony Screening
Select 5 clones to mini-prep, digestd by Kpn I and Xho I. 373-1,-2,-3,-5 may be correct. Control is 373-1.
pDonr221_EGFP(393) + pAd5-CMV-Dest-Flag(373-1) make gateway by LR Clonase II.
Transform 393+373-1 by STBL3, Amp.
Select 4 clones to mini-prep.
373+393 1-4 plasmids are digested by BamHI. Control is 373+393-4.
373+393 -1 and -4 may be correct.
2. Cloning of pAd5-CMV-Dest-Flag (373')
Purpose: to clone pAd5-CMV-Dest-Flag by PCR
Date: 2011-12-16 to 2012-1-3
Notebook: Xiaojing Wang
Page: 004127---004131
Clone ID: 373'
Experimenters: Xiaojing Wang
Results
Primer:
ad-GW-F
Kpn1
aactc GGTACC ATCAACAAGTTTGTACAAAAAAG
ad-GW-Flag-R2
Nhe Xho1
(cttTCTTGTACAAAGTGGTTGATGATTACAAGGATGACGACGATAAGtga gct agc TCGAGatcga)
tcgatCTCGAgctagctcaCTTATCGTCGTCATCCTTGTAATCATCAACCACTTTGTACAAGAaag
Digest result:
Ligation, Transformation
Select 5 clones to mini-prep
373' plasmids are digested by Kpn1 + Xho1, 373'-1,-2,-3,-5 plasmids may be correct.
pDonr221_EGFP(393) + pAd5-CMV-Dest-Flag(373'-1) make gateway by LR Clonase II.
Transform 393+373'-1 by STBL3, Amp.
Select 4 clones to mini-prep(i.e.373'+393 1-4 plasmids), then transfect 293T cell line.
48 hours after transfection, take pictures.
393+373' 1-4 plasmids are digested by BamHI.
373'+393 -1,-2, -3 and -4 may be correct.