gRNA kits say to "linearise" the plasmid containing your promoter+gRNA right after the termination signal. I was wondering whats the point of having the whole backbone there?. Cant you just cut at each side of your cassette (Promoter+gRNA) and electroporate that into your cells? Some of these kits say to use 4ug of template per reaction which it sounds to me like a lot. In addition, 4ug of a 10kb linearised vector containing your Promoter+gRNA wont be the same as transfecting 500bp of promoter+gRNA (i.e. without backbone). If you can cut at each side of your cassette and use this for synthesis cant you probably get away with using 1ug only or less?
On a different note, could you PCR with a proofreading polymerase your promoter+guides and transfect that? I find much easier to get large amounts of DNA this way. My only worry is that not 100% of the copies will be error free and if you are doing point mutation genome editing you may want to use a plasmid?
Any thoughts with these questions would be highly appreciated.
cheers
Hi Patrick,
you could do pretty much along the line you have described. The only precaution - you shouldn't cut to close to the promoter and 3' termination, couse linearized DNA will be digested by endogenous endonucleases. So to have some room around helps to sustain expression full length. Foe the same reason some guys do prefer transfecting circular plasmid. PCRing is fine too with the same precautions ( some room around) . With pfu you will have less than 1 mutation per 1 kb so with 3 kb product just 0.3% will be affected and most will be simply shorter, inactive products. So you could use PCR pretty much as plasmid. My guess is that people use cut plasmid instead of PCR, couse it easer to get high concentration fragments for transfection without additional steps..
- Correction: The accuracy of Pfu DNA Polymerase has been reported to be 7.7 × 10^5 to 1 × 10^6 (Promega). So taking the lower value, say, 10^5 - 1 mutation per 330 x 3kb fragments gives 0.3 % affected.
Hi Dmitry,
Many thanks for your response. Hope you don't mind me asking, but I was wondering if you know roughly how much space do you leave? is 10bp ok? or are we taking on the 100s?
I find when I digest from my 14kb plasmid the 300bp worth of promoters driving 2 gRNAs that my 300bp band is very very faint, even when I carry out a digestion with 3ug DNA. I have done this same digestion in 2-3 large lanes and pooled the DNA for gel purification but I find this amount is very unlikely to be enough for the RNA synthesis reaction which recommends using ~4ug DNA template. However, I wonder if 500ng of 300bp fragment would be just as good as using 4ug of the linearised plasmid given the difference in size. Has anyone tried this?
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