First of all, there are some considerations.

1. What type of polymerase you are using ?

2. What are the melting temperatures of the both primers ?

The problem here I see is the elongation is not proper since you have a small product or the template is contaminated. I would suggest to you new set of primer dilutions (ideally 10 microM from where take about 1 microliter to the reaction mixture) and also make fresh high quality dNTPs.

If you are using Taq polymerase, I do not think 1 min elongation is enough. Usually the rate is 1000 bp / min but there are high chances of a recessed product. So, I would suggest to at least give a 2 min elongation for 1500 bp product.

If you are using a high fidelity polymerase, I would recommend going +3 deg higher than the lower Tm. This is quite stringent and then give an elongation time of 30 secs, since they have a rate of 1000 bp/ 15 s.

Tell me more about it then i can help better.

Note: I also agree with Rajshree about running a gradient to observe which temperature gives you good product but please use 2min elongation in this case as well.

Regards,

Sudip

Hi, where are your primers from? Out of a publication or...?

Yes, its out of publication.......

You are getting two bands with low molecular weight. Without seeing your gel or knowing the sequence of your primers, my guess is that you might have some non-specific annealing of primers to your template. What is the melting temperature of your primers?

Your question does not contain enough details to make a good recommendation. In general non-specific products are formed when conditions are not stringent. To easily test the optimal conditions for your reaction, I recommend you use a strategy to optimize the conditions (PCR machine with a gradiant block capability). I also recommend doing some upfront bioinformatic analysis on the primers you are using, the prescribed buffers, and assess whether the design is appropriate for your objective. For instance, not all polymerase activity will be optimal under some conditions, potentially resulting in truncated products. Good luck!

Hi,

you should try gradient PCR to set the real conditions if not so then use a negative control (no template) in your PCR to check whether your own samples have contamination. then try with new DNA or cDNA.

bye

I work with PCR samples of 1,500bp. They are suppose to show one single band even for a broad community of bacteria. My guess is that you have contamination. Set up negative and positive controls to your experiments. The answers above are all valid and they are pretty much explaining what is happening.

@Gisella,

I don't do PCR with products as large as 1,500 bp, but I thought the rule of thumb was 1 minute/kb. Do you use a 1 minute extension time for your successful PCR runs?

When you have your primers out of publications, kindly check that publication for other specifics, eg. Magnesium chloride conc., melting temp for primers, etc. Also I recommend use of high fidelity DNA Polymerase enzyme. So first of all, understand your primers better, coz it may be a contamination case or non specific amplification that you are facing.

Get hold on primers, standardize using a gradient PCR with proper annealing time and temperature and good quality polymerase. Good luck!

Thanks to all of you for your kind suggestions i will consider each and every of them...

First of all, there are some considerations.

1. What type of polymerase you are using ?

2. What are the melting temperatures of the both primers ?

The problem here I see is the elongation is not proper since you have a small product or the template is contaminated. I would suggest to you new set of primer dilutions (ideally 10 microM from where take about 1 microliter to the reaction mixture) and also make fresh high quality dNTPs.

If you are using Taq polymerase, I do not think 1 min elongation is enough. Usually the rate is 1000 bp / min but there are high chances of a recessed product. So, I would suggest to at least give a 2 min elongation for 1500 bp product.

If you are using a high fidelity polymerase, I would recommend going +3 deg higher than the lower Tm. This is quite stringent and then give an elongation time of 30 secs, since they have a rate of 1000 bp/ 15 s.

Tell me more about it then i can help better.

Note: I also agree with Rajshree about running a gradient to observe which temperature gives you good product but please use 2min elongation in this case as well.

Regards,

Sudip

Hi,

I have somehow the same problem: I want to amplify 16Srrna gene with GM3-GM4 primers (1500bp) and for a couple of days a get amplicons at 500bp and 400bp, besides the wanted one. I varied the elongation time (from 1 to 3 min), the annealing temp. (49 to 52oC), and also I try changing the reaction components. I use Tag poli. The strangest part is that the negative control is always ok (no contamination).

Also check for mispriming.

Hi, did you check your DNA concentration as well? Cheers, Nadine

No, because I used cell pellet directly.

I’m doing a gDNA extraction today and I hope that this should be the problem although it was going well until now.

Hi,

Definitely follow what other people have suggested. But check also the specificity of the primer pair. You can do a Blast with your primer sequences and see the result. Good luck

Sreemanta

Sounds like a primer specificity problem. Try using the tool Primer BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) to check.

Tanks! Problem solved. The primers were the problem. Although GM3-GM4 primers are ok for my strains (P. fluorescence), this time -for a very closely related Sp. -, primer pair 8 (f) -1492 (r) have proven to be better.

Thanks for everything!

And I hope my intrusion helped you to Muhammad.

Hi Muhammad,

I Agree with primer specificity or contamination. To test for contamination run a no template reaction as a control. If that's clean and shows no product move on to troubleshoot primer specificity as suggested above (gradient PCR or redesign primers).

What concentration of template you used???

You check your primers by bioinformatic tools and recheck their difference of temperature.change concentration of primers and dNTP as well.

have a good luck

I had a similar problem and changed polymerase to another make. molecular biology is very intriguing as you never know what you have beforehand and what you will get.

Dear ilyas,

its primer specificity problem, so need to just blast your primer sequences in NCBI Blast and check its homology, if it have homology with more then one sequences (especially with last nucleotides of primer) then its give more then one amplified products and other way go for gradient PCR with low annealing temperature, hope its work

Check ur Template concentration and look out for ur polymerase.

Increase annealing temperature, reduce cycle number. if all fail, redesign primers.

epicentre company has good pcr reagents. They have a kit that has 12 buffers that you can try. One of them is going to work for you. We routinely use it for our difficult PCR.

Hi my friend

You should just blast your primer sequences in NCBI Blast and check its homology.

Good luck

I think, first, you do gradient-temperature in PCR. if it didn't resolved, reduce DNA concentration or Primer concentration.