Hi,
While extracting arctic char (Salvelinus alpinus) DNA from muscles, fin clips or gill rakers (the storage of some of them may be questionnable), I recover very low DNA yield compared to extractions that has been done on the same samples a few years ago.
The extraction protocol is slightly modified from the one of the Wizard SV 96 genomic DNA purification system (Promega).
Any ideas of what could fail in the protocol, based on your experience?
Alternatively, do you have other methods or tips that I could use to obtain a high yield of DNA from fish scales extractions?
(Attached a picture of one of the 0.8% agarose gels)
Many thanks,
Noé
I don't think you will get any satisfactory answer for your question. Anything can go wrong with the kit based extraction. You need to choose right kit and purification modification according to your samples and also cross check with similar work or kits you might have used earlier. I would say DNA extraction is not that hard thing as your gel shows. I barely see any DNA on your gel, so there was something very wrong with your method.
Improper storage of the samples may be the reason for this low yield.
if your yield is low then it may simply not be visible on a gel if it is smeared over a wide size range. The important thing is how much you have in terms of how many pcr reactions you can do. Are you measuring the amount on a nanodrop or similar machine and can you run a successful pcr on the dna and how many pcrs could you get from the purification and is that number enough for your study?
Many thanks for your answers!
Abhijeet Singh : I agree with you, the issue could come from many different things. The crazy thing is that my lab has been using this method successfully on other samples during many years!
Sandeep Kumar : This is one of my main concerns. Although, I have extracted DNA from scales with the same method, and still had no DNA.
Do you know tips or tricks to obtain higher amounts of DNA from scales?
Paul Rutland : I agree with your comments. I didn't measured on Nanodrop yet but it is certain that the amount of DNA is way lower than expected. I want to develop SNP markers with a ddRADseq, so I need DNA of good quality but also in high concentration (ideally 50ng/mL).
I usually prefer phenol chloroform extraction method and that gives me very high yield I have no idea about the kit you mentioned but just as a check to do you can probably increase the sample size initially and reconcentrate it using whatever buffer or water you are using
Some of the causes of low DNA conc might be as a result of less sample used for DNA isolation, or faulty extraction kit, you can do troubleshooting using separate sample extracted before, quantify your extracted DNA using nanodrop, you can also check the expiry date of your agarose.
I see that you are using the wizard sv purification kit. This has a size cutofff at about 10kb and is mainly used for pcr and gel fragment purification. A genomic dna purification kit may work better but if this kit really has been working for you in the past you could try using a double volume of hot (70c) elution buffer in case the dna is still bound to the matrix
Dear Noé Barthelemy
I opinion that Improper storage of the samples may be the reason for this problem.
Good luck.
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