We are trying to extract DNA from different wild bees. All individuals are collected with same methodology (pantrap), then preserved in Ethanol and then dried and mounted on pins. Material was collected during 2013, 2014 and 2015 vegetation seasons. We do extraction with same protocol for all individuals, but for instance in the 12 samples 2-3 works fine (we have good band) and others don't (nothing). and this happens each time. Can anyone say what can be a problem?
Hi George,
This may have several reason.
The two major ones (based on our experience):
1. The liquid in the (pan)trap. I do not know, whether you are using any kind of 'killing' chemicals there, but many of them (e.g. chloroform, glycol, ...) cause a quick degradation of DNA. The freshly collected (or right after trapped also removed) individuals may still have some 'working' DNA.
2. I do not know what kind of protocol you are using, but if you have any ethanol residues in the extracted DNA it will prohibit the PCR reaction (I do not know, how do you check the presence and quality of DNA).
By the way, your experience/results might be interesting for us as well. We are doing something similar here.
Best,
Ferenc
Why don't you try to extract DNA when they are in ethanol before mounting them on pin? You can use non-destructive methods for DNA extractions and then you can mount them on pin.
Bad reagents, primer design, thermocycler parameters, or nonspecific binding to other template sequences could be the cause.
Also when you resuspend your DNA preparation with TE buffer (10mm Tris, 1mM EDTA) EDTA is a powerful chelating agent, it is possible it can bind your Mg and thus inhibit your PCR. Also be aware that many chemicals used in the very preparation of DNA (Phenol, Chloroform, alcohol, detergents) can strongly inhibit PCR and therefore if they are carried over into the final product it may cause your PCR to fail or slow considerably.
For more information check out this very useful PCR troubleshooting document by Bernstein 2015
http://www.fws.gov/aah/pdf/pcrts1_noband.pdf
Dear Luis
Thank you very much. Your answer is very useful.
All the best
George
- Badges
- Science method