4 replies to this topic

[samm]

help!
i´m looking for the best method to dissolve dna-pellets,after it is extracted. the pellet is not completely dissolved in TE ph 8,0 and therefor the pcr shows not the results it should be. do you the best way to dissolve the dna-pellet??please tell me!!thank u..

[hula]

What method did you use for your DNA extraction? Are you sure the unsoluable pallet is DNA? What does it look like?

[kant0008]

Hi!
I agree with hula, it could be something other than dna that you've isolated. Otherwise could be a number of things:
1) Did you have an ethanol wash as the last step of your protocol? If the ethanol wasn't dried off properly your nucleic acid will not dissolve. I normally dry my samples with a pipete tip (capillary action, upside down) and then leave them for a while with lids open (cover with tissue or something) on bench or ona heating block (not too hot). With the samples you alredy have you can do an ethanol precipitation and redissolve
2) Your dna might be very concentrated and so reached the extent that it'll dissolve, what's the concentration? Try adding more buffer.
3) Leave your dna at 4C overnight to allow to dissolve, genomic dna takes a while to dissolve
Hope this helps!

[hula]

also if your DNA is high molecular DNA and looks viscid, dissolve it in 8mM NaOH solution, or 20 to 500ul to 10mM Tris HCl, 0.1mM EDTA, pH 7.5. Complete dissolution of the DNA may requires several hours of agitation at 37C or 55C (may need overnight).

[Calamhex]

If an overnight incubation in TE does not dissolve the stuff that you see... my best advice is to remove that stuff, which is probably just junk by centrifuging and transfering clear supernatant to a tube and then retry your PCR. The junk may contain PCR inhibitors...
Also, sometime with DNA dissolved in TE one has to slightly increase MgCl2 concentration... usually 0.25 mM is enough... depends on volume of DNA you are adding though...

HTH