20-119ng/ul of DNA is more than enough to get an amplification. I guess the quality is ok too? Thus if it is not possible to go to the field again I'd use a good polymerase (e.g. HiFi) and for example add DMSO at 5% before running a temp gradient for a 35 cycle PCR that has worked previously.

Just a control question, but your also running a positive control? is the problem really the DNA? Also why do you think your DNA is degrading, how are you storing it?

Thank you Sir for your help. I am storing in -20 C but we faced several hours current off. Nanodrop reading shows than that of previous reading i.e., reducing amount of template. I am using universal primers of ITS by White et al. can you Sir kindly give me the PCR cycle. Sir, what is positive and negative control?

First you should conform the failure is due to degrading of template DNA or due to disfunction of other PCR agents, i.e. buffer, dNTPs, Enzyme and primers. Try a PCR with all new agents or agents that other people used fine and the same thermal profile as before. Do run a positive control with a template you are sure to work.

As Russell said the quality of DNA should be OK. I met such a situation before, and it turned out that my primers was degraded.

Hey Chengmin Shi, Today I used new bach of Taq buffer and Mg and I added 1-1.5 µl of template DNA and 1.1 µl of primers each. Is the primer concentration is less? I load 5 µl of PCR products and got nothing but primer dimer. Are there any problem with primer and template conc.??

1 st pic with raw DNA template(1-1.5µl) and new batch of Taq buffer and Mg, dNTPs

2nd pic Previously diluted DNA template.

With that cycle I have tried, but no result.

How to understand that my primers got degraded because I am getting primer dimer

Hi Biswajit, you seem to get product in the 3 and 4th lane. For optimizing PCR condition, remember changing only one agent in a single reaction. You can still get primer dimer but no product if the last base of primer was degraded.

P.S. positive control, a reaction using template you are sure to bring product.

negative control, a reaction without template.

But if your not running a positive and negative control is your result from 2nd pic real or due to a contamination? I guess you have sequenced, but you should always have at least a negative control (H2O), and a positive control when possible.

But if your PCR has worked previously and the product is correct, and not contamination, then I'd use the same protocol and check reagents (optimize), as suggested.

Looking at your pic you are using to much primer, if you are running oligos specific to the template then 0.5ul of each primer in a 25ul reaction is more than sufficient (1ul with degenerate primers). But to check (in separate reactions) and get an answer to your problem you really need a positive control (dna from another plant that has worked with these primers)!

Check for optimization:

1. primers

2. dntps

3. polymerase

4. buffer

Though from experience and as previous, I'd throw away the cheap taq polymerase and buy a real polymerase, try 5% dmso in a temp gradient on a positive control before running your own samples.

Also nanndrop simply measures nucleotides, to get an answer as to the quality of your DNA you could either run it on a gel or use a qubit, which will measure dsDNA.

regards

Yesterday I got amplification in all samples but along with that I got amplification in negative control too. I took everything from new batch but not the MiliQ water. I am trying to amplify the matK gene. Now my question is Is fungus are having matK gene too like plants? So, where from it got amplified?

Your amplification in the negative control negates any results from that batch of samples. You should make sure a) your equipment is decontaminated (pipettes, tips, tubes, etc.), b) make sure you use all new chemicals (everything here, not just water, etc.). and try again. As a previous answer states, you should make sure you have a positive control in there, something you know works every time, along with your negative control.

After you remove your contamination and are sure its gone, one possible suggestion is that you might have too MUCH DNA in your reactions and could try a dilution curve on those. This on top with the temp gradients, changing Mg concentrations, etc. are troubling and take time, but should help you find your answers. Good luck! -a

Dear Sir, after that I checked with negative control and now its fine. I am not getting anything in negative control. It was because of water. I got amplification in case of matK (approx. 1000 bp) but not ITS2. why? Is ITS 2 and 1 are difficult to amplify?? I am using the primers originally developed by White et al.

There are a large number of factors that could play into this. Your temperature program could be off (try running a thermal gradient). ITS could require more Mg than matK for your species of interest. Your primers could be "bad". I would suggest making a list of things to try and test for ITS and then systematically going about them one by one until you fix the problem. Remember, only change one thing at a time and always use positive and negative controls.

Congratulations Biswajit! Your DNA template is fine.

Follow Andrew suggestion I'm sure you will figure out the problem. Anyway all the things above make me suspect your primer quality. DHPLC can check the primer quality but you may not accessible. Ask the company how the primer was purified, and for a quality report might be of some help.

The ref for ITS primer should be:

White TJ, Bruns TD, Lee SB, Taylor JW. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols: a guide to methods and applications. San Diego, California: Academic Press.p 315–322.

but you have to check the primer sequences to confirm the authorship.

It just occurs to me, the universal primer often have some degenerate sites which might cause poor performance for primers in some situation. It is worthwhile to design taxon specific primer.

Good luck!

Thank you very much Sir. Actually, yesterday, I have checked with low and high template concentration and comparatively high primer concentration. I didnt get any result but primer dimer. I have ordered new batch of primers from IDT (desalted) but previously what I was using of HPLC purified. I am totally demoralized.