Difficult DNA extraction-collagen mutant nematode refusing to give up its DNA - (Jul/12/2018 )
We are having HUGE problems extracting DNA from our nematode (not C. elegans). It is usually very easy to extract DNA from these nematodes using methods developed for C. elegans. However, we have used CRISPR to introduce a specific dominant mutation in a collagen gene. The nematode is exhibiting the expected phenotype and the phenotype is inherited through many generations. However, we can't extract DNA from any of the mutant lines using many different protocols. All the protocols have worked well on wild type controls. The last thing we need for the paper is to sequence the gene to show the mutation.
So far I have tried the single worm PCR method (based on proteinase K digestion), trizol, grinding in an extraction buffer then trizol, the puregene kit from Qiagen, extracting the gonad (thus removing the cuticle which has a lot of collagen in) and trying the single worm PCR method on the gonad alone. I have never been able to amplify either the gene in question or a unrelated myosin gene (which I am using as a positive control in case the collagen gene was deleted or disrupted) using several different primer sets, suggesting that I am either not extracting any DNA or that the DNA has significant inhibitors in there. Diluting the DNA has not worked either.
If anyone has any idea how to solve this we would be very grateful.
It sounds like you have tried all the usual things, and your mutant collagen may be the problem. I have no idea how to deal with it, but I did some digging the around the internet reading about the properties of collagen and found an article about the enzymatic degradation of collagen for the production of gelatin (for the food industry). It talks about enzymes other than just Prot K, so see if there are others you could try. Maybe a cocktail of enzymes. It would be a real experiment; with no idea whether it would work until you try it.
Thanks, I like the idea of a cocktail of enzymes. I have been toying with the idea of a collagenase of some kind. I will give it a try.
How did you did the grinding? with a dounce homogenizer?
How did you performed the proteinase k digestion?