I use commercial kits for extraction of plasmid DNA. But i don't have high plasmid DNA. For ex. i have tried MN (Macherey Nagel) NucleoSpin® Plasmid, Zyppy™ Plasmid (ZymoResearch) kits and got just 50 ng/ul, 70 ng/ul DNA from 10 ml culture, respectively.
Can anyone suggest another kit or method?
Sir, I think you should use Promega kit. Attached is the protocol. All the best.
If you're getting poor results from multiple plasmid prep kits, the kits may not be the problem. (That being said, I've always had the best results with MN kits, the most irregular results with Invitrogen, and the most consistently acceptable results with Qiagen.)
Here are a few other things to check:
Are you isolating plasmid from a bacterial strain with nucleases? Additional wash steps may help. Even without nucleases, additional wash steps may increase your yield.
What kind of ethanol is in the precipitation buffer? Some of the manuals say that 95% ethanol is fine but 100% may work better.
Are you starting with too few or too many cells? For usual E. coli lab strains, 5 to 10 ml of an overnight culture is usually fine, but less than that will yield less plasmid DNA, especially if the cultures aren't growing well for some reason. Too many cells may lyse poorly or clog the filter (that probably isn't the problem here, I'm guessing).
Finally, you can see what kind of results you get from a simple protocol like the one I've linked below.
Good luck!
http://www.bioinformatics.nl/molbi/SimpleCloningLab/miniprepping.htm
Hi Inci,
you don't say whether your plasmid has a high or low copy number.
If it is low then the quantities you are getting might be consistent for the plasmid.
If it is high however one possibility is that you have too much DNA. Adding too much DNA to the columns results in poor yields. I suggest you try using less culture 1-2mls is plenty or loading the cell lysate onto several columns.
-Richard
You need to establish the amount of available plasmid DNA you are trying to isolate. Is this a high copy or low copy number plasmid, you may want to try enriching for the plasmid by growing in the presence of an antibiotic if it is a suitable selectable marker of your plasmid. Consider if your lysis step is sufficient for the organism you are working with. Perhaps try varying amounts of biomass, since you may be overloading or underloading the column. If you would prefer to try a manual method, the alkaline-lysis method is excellent (the basis for most kit based extraction methods).
We have always used Qiagen kits for isolation of plasmid DNA with good success. (https://www.qiagen.com/us/shop/sample-technologies/dna/dna-preparation/qiaprep-spin-miniprep-kit?cmpid=QvenGA3Qiaprep_brand&kwid=qiagen+miniprep+kit ). We have also utilitzed their maxi kits in the past as well. For low copy plasmid I would suggest NM as well.
In addition to available kits, the alkaline lysis method by NaOH, SDS is also effective and gives excellent results. Please refer to Molecular cloning by Sambrook et al. for detailed protocol.
I use a miniprep kit from Jena Bioscience. It always works fine and is not expensive. It comes with everything including the protocol.
Good luck.!
We use the fermenta's GeneJET Plasmid kit(Cat. # K0503) and we get amazing results.
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