I have tried purifying a fluorescently-labelled (Alexa647) PCR product from agarose gel using the Macherey Nagel Nucleospin kit but even after several elution attempts (reapplying the eluted volume, heating to 70C, all the recommendations by the protocol to increase DNA yield), I can see that the DNA is still bound to the silica membrane in the column (by imaging the column with Alexa647 settings). I guess the fluorophore may be bound more strongly than unlabelled DNA to the silica membrane?
Does anyone have any suggestions for either: elution of fluorescently labelled DNA from the column or a column-free PCR/gel purification protocol?
Karandeep Kaur
Thanks for your answer. Unfortunately, this kit also uses silica-based membranes, which my PCR product seems to bind very strongly to. The problem is the elution from silica membrane columns
That's interesting.
I have not come across this. I have two suggestions.
1) Try a variety of elution buffers:
ddH2O
TE-Buffer
Maybe low pH (acetate, pH 3.6, neutralise immediately after elution)
Maybe high pH (bicine, pH 9.0, neutralise immediately after elution)
2) Use a different method:
You could attempt electro-elution
https://cshprotocols.cshlp.org/content/2006/1/pdb.prot4023.full
A method for the recovery of DNA from agarose gels. - PMC (nih.gov)
Article A method for the recovery of DNA from agarose gels
or,
Precipitation
How do I perform a DNA precipitation to concentrate my sample? (qiagen.com)
https://www.qiagen.com/us/resources/faq?id=5d591b8b-968a-4a17-849f-9d0f719b40af&lang=en
3) Bonus suggestion - don't purify it, take the amplicon product and use it straight away for your next operation, or, better yet, use it as a template for a further PCR using non-fluorescently tagged dNTPs and make a 'normal' non-fluor tagged amplicon which you can then purify using a standard nucleospin type cleanup.
Best of luck!
Hopefully it is still useful following your purification if it has the fluorophore...!
I'm not familiar with all of these new kits for purifying things but if your labeled PCR product is in an agarose gel, would it be possible to electrophorese the product from the gel into a dialysis bag?
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