Hi all,

I am doing an SSR project on Pecan nuts and I am struggling to get amplification on my PCR. Here is a summary of what I have done so far

1. DNA extraction was a modified CTAB protocol (Porebski et al., 1997).

2. Reagents were all made up fresh

3. DNA was eluted in 200μl of TE buffer, DNA was visualised on a 0.7% Agarose gel

4. For PCR a Dilution factor 1:1 was used

No PCR products were visible on the gel

5. Extracted DNA was subsequently purified through extra Surefood columns kit and visualised on 0.7% agarose gel

6. DNA appeared cleaner on gel and brown colour no longer present in the elution buffer

7. Test PCRs were set up using newly purified DNA (using 1:1 dilution and DNA straight to find suitable concentration)

8. New working stock was made of the primers.

No PCR products were visible on the gel