Hi everybody. I bought a gene synthesized (1000 ng lyophilized) What concentration do you recommend for work with it?
Hi Jefferson,
synthetic genes are usually shipped cloned into vector plasmids suitable for cloning in E. coli. You should transform it in E. coli so that you can biologically amplify it and then extract huge amounts of plasmid for further use.
Anyway if you just want to amplify you synthetic construct by PCR, you can do it by using serial dilutions of your template, starting from 10 ng, down to 0.01 ng. The amount of template to be used depends on the molecular weight (and hence the number of copies) of your construct, usually a normal PCR reaction can easily amplify as few as 100 copies of template. Make sure that your primer pair works fine.
Hope this helps,
Francesco Santoro
Hi Jefferson,
synthetic genes are usually shipped cloned into vector plasmids suitable for cloning in E. coli. You should transform it in E. coli so that you can biologically amplify it and then extract huge amounts of plasmid for further use.
Anyway if you just want to amplify you synthetic construct by PCR, you can do it by using serial dilutions of your template, starting from 10 ng, down to 0.01 ng. The amount of template to be used depends on the molecular weight (and hence the number of copies) of your construct, usually a normal PCR reaction can easily amplify as few as 100 copies of template. Make sure that your primer pair works fine.
Hope this helps,
Francesco Santoro
I agree with Mr. Santoro. A serial dilution of your DNA ranging from 0.01 ng to 10 ng should be good starting DNA amounts for PCR reactions.
Liza Robles
Hi Jefferson
I agree with Francesco. You should first check if your gene is cloned in a plasmid. When I ordered a few synthetic genes they came in Topo vector. So I first transformed in e.coli so I wouldn't lose my DNA.
Thanks everybody for your answers, I tried gradiente PCR with differents concentration and I got fine between 0,01ng and 10ng
Hi Jefferson
I agree with Mr.Francesco. I think the crucial step is to amplify it by inserting it in a vector( if it isnt so). Afterwards you can try three different concentrations between 0,01ng and 10ng .
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