Previously for genotyping from mouse tissue, I have used an extraction method which involved SDS-proteinase k, followed by RNAse A and Isopropanol, then resuspension of remaining pellet in buffer for PCR. We never had an issue with amount of DNA with this method.
My current lab uses a phenol-chloroform extraction, which seems like 'overkill' for DNA extraction for the purposes of PCR and microsatellite analysis. Plus, there are the health and environmental impacts of using these materials.
Has anyone tried both methods - are there additional drawbacks or considerations to think about?
Enzymatic method
Pros - clean, easy
Cons - expensive, cmparative lower yield, inhibitors might still be present
Phenol-chlorofom-isoamyl method
Pros - cheap, higher yield, removes inhibitors, most popular
Cons - needs more expertise, longer duration protocol, significantly hazardous
I have sued both
for extraction of genomic DNA at one point it was common to perform SDS Prot K digestion followed by phenol chloroform followed by isopropanol (1 volume) or ethanol ppt (3 volumes)
The advantage of this is that your samples are more effectively deproteinated so cleaner DNA
Disadvantage: Loss of DNA and use of hazardous volatile organics meaning it has to be done in a safety cabinet
In reality people have found that simply ppt crude DNA is enough in general for genotyping PCR to work with validated primers
This therefore is quicker and less hazardous
The disadvantage is that for new sites (primers) which are not optimised it doesn't always work as reliably as phenol treated material
Thus, in general simply ppt with 1 volume of isopropanol
If however you experience poor signal for that subset of samples you organically treat before ppt
The tiny amounts of phenol used are something of a overly feared thing. I use a chemical fume hood to work under, really it isn't that big a deal. I have seen lab managers in full pants-s****** mode over a few hundred ul of phenol solution, treating it like it was plutonium. ridiculous.
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