DNEasy Blood and Tissue Kits

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flyfish
flyfish's picture
DNEasy Blood and Tissue Kits

My lab has been attempting to use the DNEasy Blood and Tissue kit for DNA extraction from single flies.  We are consistently getting much lower DNA yield compared with the labs usual, rougher, method.  We need to use the kit because it produces purer samples, and these particular extractions need to be clean.  We cannot increase the amount of starting material.  We have attempted to modify many aspects of the protocol to increase yield - longer incubation for lysis, etc. and nothing seems to be working.
Does anybody work with this kit and have experience trying in increase yield?

Ivan Delgado
Ivan Delgado's picture
 

 
Hi flyfish,
I've used the DNEasy tissue kits a lot and this is what I can tell you: assuming that the column for some reason does not react in an adverse way towards your fly lysate (unlikely but possible), in other words it just does not bind your DNA that well because of the lysate composition, you should be able to increase your yield. One way that I have gotten higher yields in the past (not encouraged, but it works) is to use warm elution buffer. Heating the buffer up to 55 to 65oC can increase your yield. Alternatively you can elute with water (in which case you should heat the water up) and perform at least two subsequent elutions. While the first elution typically elutes most of the DNA, by combining multiple elutions you can significantly increase your yield (and because it is in water you can then concentrate the elution). 
A minor point too is that you need to make sure that you are completely lysing your fly every time you extract DNA. With these kits that tends to be one of the main sources of lower yields. 
Good luck
 

R Bishop
R Bishop's picture
Flyfish,

Flyfish,
Perhaps you can produce DNA by your "dirty" method and the clean it up using a qiagen spin column like those found in the DNEasy kit.  Ive done this many times to clean up DNA for injection of plasmids into mouse oocytes?
 
Rus