Problem with amplifying DNA from FTA cards - Is there a special trick involved? (May/25/2005 )
I've been trying to amplify DNA from FTA card punches for a little while now, and so far have had zero success.
I'm using Whatman FTA cards, some with bovine semen, some with bovine blood. I've even run a test where I put purified DNA onto one of the cards. None of them have amplified.
I'm using the standard protocol provided by whatman. I take a punch, wash it for 5 minutes twice in the Whatman purification reagent, then wash it for 5 minutes three times with TE buffer. I then dry the punch in an open thermal cycler (don't have a drying oven) at 40C for an hour. From there, I proceed to add my PCR mastermix and go through my normal PCR procedure.
My positive controls using purified DNA (no FTA card) work just fine. I'm running a gel of purified DNA added to a blank washed FTA card punch today (just to see if there is some sort of inhibitor in the card).
Does anyone here have experience with this sort of thing?
I've determined that an extended initial wash step (~60 minutes) using DTT helps increase yield. However, it seems that FTA cards are not anywhere close to purified DNA in terms of reproducibility. Where one would get 99% PCR success with purified DNA, it appears one can expect around 75% PCR success with samples from FTA cards.
FTA cards are convenient for long term storage, but they are quite labor-intensive to use.