1758 pCMV5-hIgG-Lc-FN3K
Cloning of 1758 pCMV5-hIgG-Lc-FN3K
[edit]Purpose: Cloning of1758 pCMV5-hIgG-Lc-FN3K
Date: 2013-6--7-
Notebook:0
Page:
Clone ID:1758
Experimenters: Shanyi
Results
[edit]cloning
[edit]PCR using
Harvard Kinase Plate HRG007900, 79
Well: G10
donor vector: pDNR-DUAL
Primers
FN3K-Kpn1-R
TGA TCG GGT ACC CTT GAG CAG CCT TCG CAT GGT G
FN3K-Nhe1-F
ATC GAC GCT AGC ATG GAG CAG CTG CTG CGC GCC GA
72oC 1min, 24cycle, Q5 HOT START POLYMERASE
Digest vector 13951395 pCMV5-hIgG-Lc-TagGFP-Fc(3c)-WPRE-OriP with Nhe1 + Kpn1
Digest purified PCR product with Nhe1 + Kpn1
ligate and transform NEB turbo
Colony Screening
[edit]Date: 2013-6-7
Make miniprep and digest with Nhe1 + Kpn1
send 1758-1tosequence, it's not we want.
cloning
[edit]PCR using
Addgene broad kinase plate#3,Well:G12
Primers
FN3K-Kpn1-R
TGA TCG GGT ACC CTT GAG CAG CCT TCG CAT GGT G
FN3K-Nhe1-F
ATC GAC GCT AGC ATG GAG CAG CTG CTG CGC GCC GA
72oC 1min, 25cycle, phusion HOT START POLYMERASE
Digest vector 1395 pCMV5-hIgG-Lc-TagGFP-Fc(3c)-WPRE-OriP with Nhe1 + Kpn1
Digest purified PCR product with Nhe1 + Kpn1
ligate and transform NEB turbo
Colony Screening
[edit]Date: 2013-6-7
Make miniprep and digest with Nhe1 + Kpn1
send 1758-1tosequence