I have buccal swabs that were collected by a lab tech in the field. The species of concern is an ungulate, if that matters.
A labmate of mine had success storing identical swabs in TES buffer (100mM Tris, 100 mM EDTA, and 2% SDS), keeping them at ambient temperature, then freezing them at -20 when he got back to the lab before he extracted them. So, I instructed our tech to follow that procedure.
I've extracted a few of these swabs and so far I'm getting virtually no DNA whereas my labmate was getting 20-40 ng/ul per swab. I can't for the life of me figure out why. Perhaps you folks can look over my protocol and offer some pointers.
I'm following the protocol outlined on pg. 37 of THIS Qiagen pamphlet with the following changes:
As my swabs are already in 600 uL of buffer, I don't add PBS.
I'm increasing the amount of Protease K to 40 uL and the incubation time to 48 hours (per my labmate's advice)
I elute in 75 uL AE (per my labmate's advice)
I'm wondering if I need to add a salt to the TES buffered swabs initially but if so then why would the extraction have worked so well for my labmate?
Should I abandon the kit and go for a phenol/chloroform extraction? Would it be at all useful to remove the swab from the TES to a tube with PBS and use the Qiagen protocol verbatum?