I isolated dried pressed herbarium plant samples using the modified CTAB method (Khanuja et. al., 1999) by adding PVP and beta-mercaptoethanol to reduce the number of polyphenols and polysaccharides. I had a DNA pellet at the end of extraction and also had a decent Nanodrop value (image attached). However, my PCR for ITS1, ITS2 (White et. al.) and rbcL (Poinar, H. N. et al.) wasn't working. Master mix of 10 µL (1 µL DNA template), including 0.5 µL (10x) BSA.
I then used the GeneClean SPIN KIT to purify my DNA using silica glass milk, and this time it worked for ITS1 but not for ITS2 and rbcL. (Image attached).
Following which I increased my DNA to 2 µL and added 0.5 µL DMSO and tried the rbcL and ITS2 PCR again. But it still failed to give any bands. (But the positive control was working.)
I am stuck now as to whether it's a DNA problem or a primer problem. What further troubleshooting must I carry out?
Since your positive control is working, that rules out a primer problem.
I know it sounds counter-intuitive, but have you tried diluting your plant DNA? Try 1:10, 1:100 dilutions. If there are inhibitors, that will lower their concentration, while still having more than enough DNA to detect. Try with just a few samples (3-4) and see if that makes a difference.
Good luck!
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