I have a problem with my sample nowadays. I am working with fecal sample preserved in DMSO buffer and has been preserved for about 4-5 years at -20C. The extraction yield doesn't look good at all. I get low DNA content and low purity, moreover, PCR doesn't run well too. What should I do to handle this problem? I really appreciate all of the answer and suggestion. Thanks.
Hello sinta,
Before isolation u should thaw the sample at room temperature .
Secondly removal of cryoprotectant that is Dmso , either by washing, centrifugation,filtration.
Filtration of sample.
Prewashing, finally isolation with black bio kit , quigen kit or shrimpex kit will yield a better DNA. Later qc and then amplify.
If still truncated DNA came cartridge purification required
Regards
Aamir javed
Hello sinta,
Before isolation u should thaw the sample at room temperature .
Secondly removal of cryoprotectant that is Dmso , either by washing, centrifugation,filtration.
Filtration of sample.
Prewashing, finally isolation with black bio kit , quigen kit or shrimpex kit will yield a better DNA. Later qc and then amplify.
If still truncated DNA came cartridge purification required
Regards
Aamir javed
https://www.qiagen.com/ye/shop/sample-technologies/dna-sample-technologies/genomic-dna/qiaamp-dna-stool-mini-kit
Proceed as Assmir Javed suggestion... Also, you could use FastDNA™ SPIN Kit for Feces. This kit gave me good results.
http://www.mpbio.com/product.php?pid=116570200&country=167
I analyze faecal samples. I using DNA-Sorb B kit (Amplisence) for isolation DNA and I have good results. After PCR (nested, real-time) and electrophoresis purified samples. DNA content is good and purity is 1.85-2.0.
Hello Sinta,
There have been great suggestions for fecal DNA extraction methods and kits. As for PCR we have to think about the quality of the purified DNA. It will be highly fragmented and high in PCR inhibitors. Regardless of the PCR target you are interested in, if you want better amplification you may want to redesign your PCR primers to flank smaller fragments (~150-300BP). If you ultimately require a longer PCR fragment, consider designing multiple mini-fragments (these will be run in separate reactions) that can be concatenated/aligned to the full fragment during analysis.
As for inhibitors and PCR efficiency, consider using an additive in your PCR mastermix, such as bovine serum albumin (BSA). 20-100ng should do fine but do a concentration gradient to find out the optimal concentration.
Best of luck!
Dan
Hello Aamir Javed,
Following your second suggestion, what do you think the best to remove the cryoprotectant? I thought by centrifugating is the easiest way than others but my college concerned about DNA floating in supernatant that will be discarded along with the cryoprotectant. What do you think about it? Then about removing it with washing do you have any suggestion the best washing agent and protocol to do it?
Anyway, thanks for the answer
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