I'm working with old and not well preserved shrimp tissue samples. My extractions have a good amount of dna (> 200ng), but with very low quality. The dna is very contamined and I couldn't amplify it. New samples are ok, but the old samples are not.
How can I purify these extractions? PEG protocol is ok? We do not have money for purification kits.
Note: I need to purify the extraction itself, not the PCR products. I want to know if it is possible to purify extraction in order to get dna with more quality, and then amplify it using PCR.
You may follow the sub sequential follow purification methods like as Phenol; Chloroform: isoamyalcohol (25;24; 1)
Hi Sarah,
We have expertise with DNA extraction from shrimp but using commercial kits.
in your case, as Daniel indicated, you should try the good old protocols involving phenol extraction an subsequent extraction with CHCl3/isoamylalcohol. Do not forget to precipitate DNA with 2 volumes of ethanol in the freezer and recover DNA pellet by centrifugation.
best regards
Hi Sarah,
You can also gel purify your DNA. Run it out on an 1% agarose gel, cut out the high quality DNA & purify. To purify, cut agarose into chunks, put in a .5 ml tube that has a hole punched through the bottom & contains a slurry of filter paper. Place this tube in a 1.5 ml tube & spin. Can then etoh precipitate. Citation (with details): Chuang & Blather 1994 Biotechniques 17(4):634-36. Long DNA may be sheared, but it still may be better than what you have now.
Or, to purify you can melt the chunks of gel in presence of NaCl, and extract with phenol/chloroform -- less shearing. If interested, I can send you the protocol (or you can probably find a published version on somewhere).
Good luck.
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