This month's questions from the Molecular Biology Forums (online at molecularbiology.forums.biotechniques.com) come from the “DNA and General PCR Methods” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but do not represent endorsements by, or the opinions of, BioTechniques.
Molecular Biology Techniques Q&A How can I get my AFLP working again? (Threads 1113 and 23649)Q1 Previously, my AFLP reactions were working well, but now—despite using the same protocol, same primers, same template, etc.—it stopped working. I can still do selective amplification for the pre-amplification reactions I did before, but no new digests, ligations, or pre-amplifications work with any of my six different selective primer sets.
I tried modifying the MgCl2 concentrations, dNTPs, etc. I threw out all of the reagents, modified the annealing temperatures, made new primer dilutions for pre-amplification and selective amplification, and increased the length of the digest. Yet nothing solved the problem, not even using the same template as the original reactions.
On my last attempt, 2 out of 3 sets of selective-amplification primers generated weak bands with a lot of background. The lower bands (70–140 bp) were visible and somewhat clean, but only a few of the major upper bands showed up at all and they were surrounded by noise.
Can someone with AFLP experience offer some insight? Have you found any component of the reaction to be particularly critical, such as DNA concentration or quality? My dNTPs were diluted many months ago, but they are aliquoted to avoid more than 3 freeze/thaw cycles. Could this have caused the problem?
A I have a similar problem with my AFLP analysis. I checked the digestion, adaptor ligation, and pre-amplification reactions on 1% agarose gels and everything looked fine. But when I prepared the specific amplification and gel electrophoresis for that step, silver staining showed no product and very strong background. This didn't change with fresh primers, dNTPs, and buffers.
A The same thing happened to me. I had my AFLP working for a month, but then it stopped. I just got it to work again by ignoring the literature and doing the ligation and amplification like any normal ligation and PCR.
This is my working protocol:
Digest 400 ng genomic DNA with 10 U EcoRI in a 40-µL total volume at 37°C overnight.
Add 100 pmol each adaptor, 1 µL T4 ligase, and buffer as required in a 50-µL total volume.
Incubate at room temperature for 2 h.
Use 5 µL ligation reaction as a template for the PCR reaction.
I am currently fine-tuning my primer concentrations for amplification.
A Are you using 33P-ATP in your selective reactions? If so, you can try a new batch of kinase.
A The enzymes in AFLP are the most critical aspects of the reactions. Each has different buffer requirements, so you must look closely to find the best compromise. I am using a homemade buffer. The recipe for the 10× stock is 100 mM Tris-HAc (pH 7.5), 100 mM MgAc, 500 mM KAc, AND 50 mM DTT. I don't know if this is better than any other buffer, but it consistently works for me.
Q2 My AFLP was running well, but suddenly stopped working. I haven't changed anything in the protocol. I checked my DNA quality using agarose gel electrophoresis, replaced the reagents, checked the PCR program, and even tried a different thermocycler. Despite these steps, the same samples that were previously positive no longer work. What else can I try to fix this?
A Your problem could be with the primers. They can get damaged if they are too old. Have you tried to order fresh primers?
Q I tried new primers, but nothing changed. I wonder if it could be due to the ligase buffer.
A Your problem could be caused by the ligase. Don't trust new tubes just because they're new. If multiple users work with your enzymes, you can't be certain how they were treated.
Q I'm the only one using the primers right now, but the enzymes are shared. I bought new ligase and buffer, but the selective amplification still failed.
A I am having a similar problem. I tried using my preselective template for several selective PCR amplifications but got no results. I re-ran samples from previous successful selective PCR reactions and got only fragments smaller than 300 bp. When I first submitted these samples about a month ago, I got bands up to 600 bp. My guess is that my preselective PCR template is degrading, which is why I'm testing that now. Could that be your problem as well?
Q I think that both the preselective amplification and ligation products can degrade with time. I preserve mine at −20°C or even −80°C if I'm not using them immediately. Do you submit your samples somewhere or run them yourself? How is the ROX signal in the cases with small fragments? Submitting samples to a facility introduces a lot more difficulty since they are out of your hands. How do you prepare the samples before sending them?
A I kept my preselective template at −20°C, but had several freeze/thaw cycles for multiple uses. I redid my digestion, ligation, preselective amplification, and selective reactions and everything worked great. I am keeping the preselective product in the fridge and plan to use it up in the next few weeks. I aliquoted and stored the ligation at −80°C.
I performed more selective PCRs this week with new primers. The results weren't the best, but they were better than before.
I send my samples to a center for analysis and only dilute to the required concentration before sending them.
A I have been doing AFLP for 10 years and have published a few papers on the technique. I can send you both my optimized protocol if you would like. The majority of problems I encountered were linked to poor digestion and amplification, so you should focus on those steps when troubleshooting.
