Hi Pratik,

You can get plasmids encoding Cas9 nuclease and a single gRNA from Addgene (https://www.addgene.org/). They have many different ones.

They send you them as a bacterial stab culture that you can grow up and isolate the plasmid from. They are far cheaper than any company selling Cas9 plasmids as they are a non-profit repository (~50-60$ per plasmid).

To clone in your specific gRNAs you use a relatively simple and cheap annealed oligo cloning technique.

Good luck.

I understand this! But for the oligo cloning technique as well, it would cost nearly the same amount as with sgRNAs so I was keen on using sgRNAs.

for oligo cloning, I have to buy oligos, then specific buffers alongwith plasmids with Cas9 and all this comes to the same amount.

To clone 4 gRNAs using a a single plasmid purchased from addgene (or free from a colleague; almost everyone is using Cas9 now). The cloning doesn't require anything special, all common buffers and reagents for a molecular biology lab.

It would cost you 25-100$- not sure you will get what you are looking for less that that!

Hi. The protocol which I will be using is this. 

https://media.addgene.org/cms/filer_public/e6/5a/e65a9ef8-c8ac-4f88-98da-3b7d7960394c/zhang-lab-general-cloning-protocol.pdf

What is the protocol you are stating?

Hi Pratik,

It is similar to what I do except: (i) phopho oligos seperately (37oC 1h then 65oC to inactivate PNK), (ii) then anneal oligos together in duplex buffer (30mM HEPES 100mM potassium acetate) cool in a thermocycler at 0.6oC/min (this is critical, it must be slow) and (iii) for ligation I use 50ng digested plasmid and use 3-7ul annealed oligos (without dilution of annealed oligos beforehand).

This has yet to fail for me and I have cloned over 60 different gRNAs constructs using this cloning.

I can send you my protocols if you wish to try it.

Yes. My email id is [email protected].

Your help would be welcomed.