I am isolating plasmid for restriction digestion using alkaline lysis protocol and getting ample amount of plasmid (1000-2000ng/ul) with very less amount of RNA residues but at the moment I use (10ug) it for restriction digestion followed by its elution (in 20ul water) I am getting 10-12ng/ul of it. I have inspected the elution kit, it is working fine. Now I am doubting the plasmid isolation method employed by me. Please suggest an appropriate solution to this. Also I am attaching an image of plasmid isolated having a nanodrop reading of 4000ng/ul whose 2ul is loaded on the gel.
Hi there,
The sample loaded onto gel doesn't seem to contain 8µg of plasmid (which is by the way far too much for a gel analysis). Also you should run calibrated DNA ladder in parallel so tha you can estimate the amount of DNA.
the gel looks more like a great deal of bacterial genomic dna and some plasmid but as Dominique Liger says it is necessary to run a dna ladder to estimate both size and amount of dna in the sample
For plasmid isolation to proceed further for restriction digestion we require a lot of plasmid DNA, at least 1ug. Plasmid isolation by Midiprep alkaline lysis is the best way to achieve good plasmid in my experience. First of all, you need to be thorough about your plasmid conc. I mean, not only nanodrop you need to quantify the plasmid by Image J software as well. At least by comparing it with DNA ladder you can have an estimate of plasmid. Next time what you can do is, Load ladder and your plasmid and quantify your plasmid with Image J and then check for nanodrop readings. Image J gives maximum approximate of the conc. present in the volume you have loaded which can be compared with the ladder. Kit based methods gives really good plasmid but always a crude method is the best.
Shifa.
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