326 pAd5-CMV-Dest-HA
The primer-Rs of 326 and 326' are different.
1. Cloning of pAd5-CMV-Dest-HA (326)
Purpose: to clone pAd5-CMV-Dest-HA by PCR
Date: 2011-11-28 to 2011-12-
Notebook: Xiaojing Wang
Page: 004127---004131
Clone ID: 326
Experimenters: Xiaojing Wang
Results
The middle bands show PCR product of 326,
Primer:
ad-GW-F
Kpn1
aactc GGTACC ATCAACAAGTTTGTACAAAAAAG
ad-GW-R Xho1
(TCTTGTACAAAGTGGTTGATtacccatacgatgttccagattacgcttgaCTCGAGatcga)
tcgatCTCGAGtcaagcgtaatctggaacatcgtatgggtaATCAACCACTTTGTACAAGA
Digest result:
digest PCR product and vector with Kpn I+Xho I,run gel
the left picture shows 326 insert, the right picture shows 326 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. 326-1,-3,-4,-5 may be correct. Control is 326-1.
pDonr221_EGFP(393) + pAd5-CMV-Dest-HA(326-1) make gateway by LR Clonase II.
Transform 393+326-1 by STBL3, Amp.
Select 4 clones to mini-prep(i.e.326+393 1-4 plasmids), then transfect 293T cell line.
48 hours after transfection, take pictures.
326+393 1-4 plasmids are digested by BamHI. Control is 326+393-4.
326+393 -1, -2 and -4 may be correct.
2. Cloning of pAd5-CMV-Dest-HA (326')
Purpose: to clone pAd5-CMV-Dest-HA by PCR
Date: 2011-12-16 to 2012-1-3
Notebook: Xiaojing Wang
Page: 004127---004131
Clone ID: 326'
Experimenters: Xiaojing Wang
Results
Primer:
ad-GW-F
Kpn1
aactc GGTACC ATCAACAAGTTTGTACAAAAAAG
ad-GW-HA-R2
Nhe1 Xho1
(cttTCTTGTACAAAGTGGTTGATtacccatacgatgttccagattacgcttga gct ag CTCGAG atcga)
tcgatCTCGAGctagctcaagcgtaatctggaacatcgtatgggtaATCAACCACTTTGTACAAGAaag
Digest result:
Ligation, Transformation
Select 5 clones to mini-prep
326' plasmids are digested by Kpn1 + Xho1, 326'-1,-2,-4,-5 plasmids may be correct.
pDonr221_EGFP(393) + pAd5-CMV-Dest-HA(326'-1) make gateway by LR Clonase II.
Transform 393+326'-1 by STBL3, Amp.
Select 4 clones to mini-prep(i.e.326'+393 1-4 plasmids), then transfect 293T cell line.
48 hours after transfection, take pictures.
393+326' 1-4 plasmids are digested by BamHI.
326'+393 -2, -3 and -4 may be correct.