2277 pET28b-Cas9-Nickase
Cloning of 2277 pET28b-Cas9-Nickase
[edit]Purpose: Cloning of2277 pET28b-Cas9-Nickase==
Date: 2014-7----
Notebook:0
Page:
Clone ID:22767
Experimenters: shanyi
Results
[edit]CLONING
[edit]PCR from 2275 CAS9P
hCas9-D10A-Nco1-F ctagcccatggacaagaagtactccattgggctcgCtatcggcac GACAAGAAGTACTCCATTGGGCTCGATATCGGC
hCas9-Not1-R (CTCAGCTCGGTGgagacagcagggctgaccccaagaagaagaggaaggtgGCGGCCGCtagcta) tagctaGCGGCCGCcaccttcctcttcttcttggggtcagccctgctgtctcCACCGAGCTGAG
digest with Nco1 + Not1, clone tov12 pET28b
Colony Screening
[edit]Date: 2014_7
Make miniprep, use NdeI+BamHI to find positive clone
Cloning of 2276 pET28b-Cas9
[edit]Purpose: Cloning of2276 pET28b-Cas9==
Date: 2014-8----
Notebook:0
Page:
Clone ID:2276
Experimenters: shanyi
Results
[edit]CLONING
[edit]PCR from 2275 CAS9P
Fragment 1
hCas9-D10A-Nco1-F
ctagcccatggacaagaagtactccattgggctcgCtatcggcac
Cas9-R2 B8 ctgaggcacgatATGgtccacatcgtagtc
Fragment 2
Cas9-F2 B7 gactacgatgtggac CAT atcgtgcctcag
1 ul of 1 uM of Cas9-R4
(gacctgtctcagctgggagacagcagggctgacccc)
D L S Q L G D S R A D P
ggggtcagccctgctgtctcccagctgagacaggtc
hCas9-Not1-R
(gagacagcagggctgaccccaagaagaagaggaaggtgcaccaccaccaccaccactgctaaGCGGCCGCtagcta)
tagctaGCGGCCGCttagcagtggtggtggtggtggtgcaccttcctcttcttcttggggtcagccctgctgtctc
hCas9-D10A-Nco1-F
ctagcccatggacaagaagtactccattgggctcgCtatcggcac
hCas9-Not1-R
(CTCAGCTCGGTGgagacagcagggctgaccccaagaagaagaggaaggtgGCGGCCGCtagcta)
tagctaGCGGCCGCcaccttcctcttcttcttggggtcagccctgctgtctcCACCGAGCTGAG
digest with Nco1 + Not1, clone tov12 pET28b
Colony Screening
[edit]Date: 2014_8_
Make miniprep, use Nco1 + Not1 to find positive clone
digest with EcoRV+BamHi
