2278 pET28b-dCas9
Cloning of 2278 pET28b-dCas9
[edit]Purpose: Cloning of2278 pET28b-dCas9==
Date: 2014-7----
Notebook:0
Page:
Clone ID:2278
Experimenters: shanyi
Results
[edit]CLONING
[edit]PCR from 2275 CAS9P
Fragment 1 hCas9-D10A-Nco1-F ctagcccatggacaagaagtactccattgggctcgCtatcggcac
Cas9-H840A-R ctggggcacgatGGCatccacgtcgtag
Fragment 2
Cas9-H840A-F ctacgacgtggat GCC atcgtgccccag
hCas9-Not1-R (gagacagcagggctgaccccaagaagaagaggaaggtgcaccaccaccaccaccactgctaaGCGGCCGCtagcta) tagctaGCGGCCGCttagcagtggtggtggtggtggtgcaccttcctcttcttcttggggtcagccctgctgtctc
Purify and Join two fragments hCas9-D10A-Nco1-F + hCas9-Not1-R
digest with Nco1 + Not1, clone to v12 pET28b
Colony Screening
[edit]Date: 2014_2_14
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
Cas8-R3 D12 ctgaggcacgatGGCgtccacatcgtagtc
Fragment 2
Cas9-F3 D11 gactacgatgtggac GCC 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
