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
Lorraine Mhoswa did you get this problem sorted or do you still want suggestions?
Hi Paul. I managed to get it sorted. I changed the PCR conditions and all the markers amplified :)
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