I did DNA isolation for 4 different insect samples (species unkown), I got DNA for 1, whereas for the other three I couldn't isolate DNA, repeated the DNA isolation procedure 3 times using PCI (Phenol Chloroform Isoamyl), and even tried spin column method. Couldn't isolate DNA.
What could have gone wrong??
Possibly the problem is with the cell lysis technique to release dna from the cells, Grinding the cells may be necessary rather than just chemical lysis to release the dna in a soluble form for purification
Paul Rutland Thank you so much for responding. Yes even I guessed the same and when I repeated the DNA isolation procedures, I did use mortar and pestle and even micropestle for grinding the cell. Still,@@@ I couldn't isolate the DNA.
Can you help me up
It may be that the amounts of dna are very small, This shows in 2 ways,,,,no DNA precipitate and no dna showing on a checker gel.
DNA not precipitating can be helped by adding a co.precipitant such as glycogen,or RNA or linear acrylamide which can help to precipitate all of the dna that may otherise stay in solution/suspension.
No t seeing dna on a gel can still mean that there is plenty of dna for a pcr reaction just not enough to see on a gel. Try running some of your fail samples with primers that usually work for you in a pcr reaction.
Measure your dna with a nanodrop or other spectrometer
Paul Rutland Thank you so much. I'll do the above mentioned and share my results.
ok one more thought . If you are grinding in liquid form or when dissolving the dry ground material make sure that the liquid has some EDTA in it to mop up divalent ions so ensuring that nucleases released on grinding are not able to spoil the dna
Paul Rutland Thanks again and Apologies I missed out on one more information. I performed PCR reaction 3 times at different annealing temperatures with the appropriate primers. Couldn't get the amplified product. I thought either the annealing temperature wasn't set correctly or the DNA was not isolated.
Hence back to square one.
And yes while lysis EDTA was included.
do you have any dna that these primers do amplify? If not you have to bear in mind that the primers may be degraded. Borrow a primer set that is currently working from another researcher and a sample that can be used as a positive control in the pcr
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