I tried all the methods available on internet be it methanol extraction or using Qiagen kit. I also tried incubating FTA card pieces in TE buffer overnight at 56 C but i got nothing. Each spot is suppose to have 125 uL sample loaded.
I have the protocol on my web site its super easy: http://www.germainhugo.com/protocols/fta-paper-protocol/
To amplify genomic DNA from Arabidopsis.
Cut a small piece of leaf.
Place the leaf on FTA paper, cover with parafilm, press hard with a glass test tube to make the leaf print.
Dry sample at RT at least one hour.
Add 50 microliter of FTA reagent to wells of 96 well plates
Take one punch from each leaves and place in FTA reagent for 5 minutes.
Remove liquid.
Rinse disc twice with 200 microliter TE-1 (10 mM Tris pH 8, 0,1 mM EDTA).
Remove liquid
Add master mix and run PCR.
FTA solution (can be made as a 10X stock. Add Tween after autoclaving).
10 mM Tris pH 7.5
2 mM EDTA
Tween 20 0.1%
if all else fails you could try just cutting a small section of the paper and adding it direct to the pcr mixture using one of the polymerases that is tolerant of impurities. Some of the KAPA enzymes are very robust. Also you may not have much dna so you could try nested pcr after the first round pcr
I use a simple method to extract DNA from whatman FTA cards - I put small section of FTA in 0,5-1 ml of the lysis buffer (with guanidine tiocyanate) of NA isolation kit (e.g. Qiagene one), vortex 5 min, centrifuge 1 min (10 000 g) and take an aliquot of this buffer for direct NA isolation by this isolation kit. This buffer I take instead of the fresh lysis buffer in the isolation process and add water instead of a sample.
Good luck!
In that case, Mr. Paul Rutland, how do i make up the volume of my PCR mix since i wont be knowing the conc. of DNA in that piece of card?
- Badges
- Science method