If the whole of your 81C Tm primer hybridises to your substrate (as opposed to say a part of it, with additional potential Tm coming from bases that you have introduced as cloning sites) then there is really too much difference in annealing temperature between your two primers. Your higher Tm primer will tend to be mopped up in non-specific binding sites, so this may explain your problem. Try finding a less GC rich position (best) or shortening the primer a bit to bring the Tm down to a value below 70C.

You may try 1) increase the annealing temperature, 2) reduce the primer concentration, 3) check your buffer

Decrease elongation time?!

Try touch down PCR program with gradual decrease of elongation time and increasing annealing temperature. I once solved such a problem using this strategy.

Depends a lot by the specificity of your primers. You can try to increase the annealing temperature in order to make more specific the binding. The best would be to make a gradient PCR if you have a machine hosting a 96-well plate. There you can try 12 different temperatures of annealing and check which one is the best one for you.

Its also a good idea to:

1. Double check your primers to make sure you didn't make a mistake when designing them. This could lead to both a decrease in your desired product and an increase in others.

2. Make sure you calculated the expected band size correctly. Actually go back to the sequence from which you designed the primers and re-verify that your expected product should be 194 bp.

If your primers are fine and the size is correct, you may want to design new primers.

You have not stated what organism or primer/ PCR conditions you are using which makes a definative answer difficult

1) Check your primers to see that thety do not pick up pseudogenes. I presume you are amplifying DNA and not cDNA.

2) if you are amplifying cDNA then check that you are not amplifying up from the unspliced genomic DNA

3) if amplifying cDNA check that you do not have alternattive splicing

Generally non-specific priming and amplification occur if the primer annealing conditions are not ideal. Increasing annealing temperatures can result in more targetted /stringent primer binding. If you are using a primer with redundancy (i.e not an exact copy of the target sequence), you can however end up amplifying non target if it is present in great excess compared to the correct target. Reducing primer annealing times can improve specificity. Most people do not look at the sequence between the primers to see if has a bearing on amplification efficiency. A GC rich sequence between your primers or a potential region of secondary structure (self annealing hairpins) can influence the efficiency of specific amplification and bias it towards "non targetted" amplicons.

Ian

You can use ucsc in silico pcr to check your product with your designed primers:

http://genome.ucsc.edu/cgi-bin/hgPcr

Decrease elongation (extension) time

I would agree with Pia that the best place to start would be simple things. Increasing the annealing temperature would increase the stringency of the PCR conditions and possibly drive the PCR reaction to the desired product of 194 bases. However, if this does not appear to work, you may need to adjust the MgCl2, dNTPs or both. Either MgCl2 and dNTPs in excess can cause amplification of false products. However, I would try adjusting the annealing temperature first as the others suggested.

Have you also considered cutting out the 300 bp band and having it sequenced? Many researchers have done this to identify an unknown band.

Good luck.

thanks a lot all of you for your valuable suggestions, i will try to work according to the suggestion given by you all.... as per sum details i would like to say i have cloned a gene in to pUC 18 vector and now using specific primer for the particular enzyme i would like to amplify the gene of interest....

What you all will suggest whether i should use master mix or else i should go for individual components for the PCR amplification..... the annealing temperature i have used is in the rage of 55 to 63 since the primer set has tm value of 81 0c for forward and 65 0c for reverse primer........

If the whole of your 81C Tm primer hybridises to your substrate (as opposed to say a part of it, with additional potential Tm coming from bases that you have introduced as cloning sites) then there is really too much difference in annealing temperature between your two primers. Your higher Tm primer will tend to be mopped up in non-specific binding sites, so this may explain your problem. Try finding a less GC rich position (best) or shortening the primer a bit to bring the Tm down to a value below 70C.

try this high fidelity polymerase

https://www.neb.com/products/m0530-phusion-high-fidelity-dna-polymerase

and also Iam agree with yue keong choon..

Before you get into time and money consuming experiments, perform a PRIMER BLAST for your pimers:

http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome

Enter your primer sequences and BLAST for PCR products on Genome reference sequences for the organism you are testing. Many times the primers are NOT unique so there is a chance that they may also be 100% complimentary to other areas of the genome amplifying unwanted amplicons. I've encountered this a number of times. If so, no matter what you do, you will ALWAYS be getting unwanted product. Therefore in that case you will need to redesign one or both primers.

I think that you should check your primers again for sure they are specific primers.

You can design a new primers if possible, then increase the Tm.

Adding DMSO (~ 10%) also can avoid the non-specific products.

If these condition are not OK, you should change the enzyme, we often use Herculase.

Good luck

I agree with all who adviced to adjust the temperature and enzyme. I also think if get non specific bands why not to try to do size selection and do a second PCr for the product?

I hope I was of use,

Good luck

what is the Tm of ur reaction? Try increasing the annealing temperature to reduce non-specific amplification.

Agreed. You may redesign your primers if possible as the difference in Tm is large. you may also verify you clone with a negative control (PCR reaction with a single primer). All the best

Try qPCR. However, just check all your parameters, like primer concentration, temperature cycles (specially the annealing temperature) weight of the DNA under study, Buffers and finally the temperature of the buffer during electrophoresis.

Your main problem is the primer with a Tm of 81oC which indicates high GC content that can cause real problems in PCR. You try to redesign your primers as suggested by others and repeat your expt.

When I design primers I use the following calculation for Tm:

[(A + T)*2] + [(G+C)*4]. Then I look at the context of the Gs and Cs.

I count up the # of G/C surrounded by G/C and add that to my Tm.

I count up the # of G/C surrounded by A/T and subtract that from my Tm.

I also try to design my primer with G/C at the 3' ends of the primers. You get better annealing that way.

Ensure that you have designed your primers such that the 3' ends point toward the product you are trying to amplify.