We have been trying to extract "good quality of DNA" using different extraction methods like Column, Phenol Chloroform Isoamyl Alcohol and AFA methods and we found that AFA works better than others since it uses less organic solvents than others. But the problem is when we are trying to do RAPD PCR we were not able to get desired fragment size(>=600bp) for our downstream analysis(Microarrays) which is very crucial for continuing to the next step. Can anybody suggest what we can do to our best to get good quality of DNA??
sear Keerti, I suppose you know this work. bellow. However, keep in mind that you might have already degraded DNA...
http://covarisinc.com/wp-content/uploads/pn_010200.pdf
look at the parameters which will minimise brake of DNA and let me know the infectivity of the sample after AFA purification fro different microorganisms?
Sorry to be so negative, but I would give up. If you do a bioanalyser trace of most FFPE DNA you will see that fragments of the size you want are vanishingly rare.
We have done quite a bit of analysis on this for NGS work, and we have found that generally the older the sample, the less likely you will find fragments even approaching this size.
What microarray platform are you using? Illumina arrays have a RAPD step within them, and a FFPE repair kit exists for this that in our experience makes ~90% samples usable.
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