gRNA annealing protocol
Lyra Chang,. Did you phosphorylate the annealed oligos? I am trying to clone a sgRNA into pX459 vector using Zhangs Labs Protocol, but at the end I do ... , Reagents. pUC-H1 gRNA cloning vector. Standard de-salted oligos. Annealing buffer: 10 mM Tris pH 8.0, 50 mM NaCL, 1mM EDTA. 10X (1ml):., 1) This protocol is derived from Ran FA et al, Nature Protocols, 2013. While not identical, it follows it closely. Read that paper for details, as well as ..., This protocol distributed from Feng Zhang's lab. - Ran et al. ... 1 h. Prepare reagents for phosphorylating and annealing the gRNA oligos: ...,I started with 2 complementary single stranded oligos (my actual guideRNA sequence), and the plan is basically to anneal them together (the protocol I'm ... ,Annealing. Mix equal volumes of the equimolar oligonucleotides in a microtube. Incubate the microtube at 95 °C for 5 min. Allow the microtube to slowly cool to room temperature (should take <60 min). ,Prepare sgRNA cloning vector: Protocol for sgRNA construction (Annealing method). Digest 5~10 μg of sgRNA expressing vector (pU6-gRNA.Ppuro) with BsmBI ... ,This protocol allows you to clone oligos to generate shRNAs or sgRNAs on a small scale. ... reaction. 1ul of annealed oligo pair (or water in a control reaction). ,Anneal oligos together by boiling at 95 Celsius @ 5 min, then cool to approximately room temperature. In my experience, any protocol for annealing will work, ... ,X ul. BbsI digested plasmid from step 2 (50ng). 1 ul phosphorylated and annealed oligo duplex from step 3 (1:200 dilution). 5 ul. 2X Quickligation Buffer (NEB).
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 gRNA annealing protocol 相關參考資料
Any advice in cloning of gRNA (CRISPR-Cas9)?
Lyra Chang,. Did you phosphorylate the annealed oligos? I am trying to clone a sgRNA into pX459 vector using Zhangs Labs Protocol, but at the end I do ... https://www.researchgate.net CRISPR gRNA Expression Vector Cloning Protocol - Addgene
Reagents. pUC-H1 gRNA cloning vector. Standard de-salted oligos. Annealing buffer: 10 mM Tris pH 8.0, 50 mM NaCL, 1mM EDTA. 10X (1ml):. https://media.addgene.org Design and cloning gRNA oligo's into Cas9 vectors ... - Versiti
1) This protocol is derived from Ran FA et al, Nature Protocols, 2013. While not identical, it follows it closely. Read that paper for details, as well as ... https://www.versiti.org Option 1. Clone gRNAs into plasmids · Benchling
This protocol distributed from Feng Zhang's lab. - Ran et al. ... 1 h. Prepare reagents for phosphorylating and annealing the gRNA oligos: ... https://benchling.com Possible annealingligation problems for oligo insert?
I started with 2 complementary single stranded oligos (my actual guideRNA sequence), and the plan is basically to anneal them together (the protocol I'm ... https://www.researchgate.net Protocol for Annealing Oligonucleotides | Sigma-Aldrich
Annealing. Mix equal volumes of the equimolar oligonucleotides in a microtube. Incubate the microtube at 95 °C for 5 min. Allow the microtube to slowly cool to room temperature (should take <60 min... https://www.sigmaaldrich.com Protocol for sgRNA construction (Annealing method)
Prepare sgRNA cloning vector: Protocol for sgRNA construction (Annealing method). Digest 5~10 μg of sgRNA expressing vector (pU6-gRNA.Ppuro) with BsmBI ... http://rnai.genmed.sinica.edu. Protocol: Cloning of oligos for sgRNA (CRISPR) or shRNA ...
This protocol allows you to clone oligos to generate shRNAs or sgRNAs on a small scale. ... reaction. 1ul of annealed oligo pair (or water in a control reaction). https://portals.broadinstitute Protocols · Benchling
Anneal oligos together by boiling at 95 Celsius @ 5 min, then cool to approximately room temperature. In my experience, any protocol for annealing will work, ... https://benchling.com Target Sequence Cloning Protocol - Addgene
X ul. BbsI digested plasmid from step 2 (50ng). 1 ul phosphorylated and annealed oligo duplex from step 3 (1:200 dilution). 5 ul. 2X Quickligation Buffer (NEB). https://media.addgene.org |