HI Nisha

You will have to find out from where or which component of the PCR reaction you are getting the 'template contamination'... is it water, buffer, enzyme or even primer dilution which you are using? Try to rule out each component one by one to find out the source of the 'contamination,' replace that component with a new one and do PCR again. If you find out that 'primer(s)' are source of contamination, you'll have to make a new dilution and do your experiment.

@Douglas

'Photshop' is not 'THE BEST' method to get rid of unwanted bands...I am sure Prof. Prasher must be kidding...

Dna contamination in the negative control. In your negative control reaction, are u using dna which is not supposed to give a band or using no dna at all? If lattter is the case, then, dna contamination is likely. If its is former case, then your primers are annealing to a different area of the genome and just happen to give a band the same size as desired band...

U can try using fresh aliquots of all the pcr reaction components. If you can use a fresh aliquot of primers, lot of times, that solves a lot of issues. Hopefully u have a master primer dilution kept separate from the working dilution.

There is probably contamination in one of your reagents that you use for PCR. To check for this, please use neat molecular grade water as your negative control. If you see a band, then start by replacing each of the other reagents with a similar one. IF this does not work, then look at the primers! They could be having some non-specific binding (very rare indeed with neat water). Thanks.

The bands are due to contamination. Do all you can to work in and around a sterile environment - buffers, enzymes, diluents, water etc. Change gloves in between. Good luck

Is the band you see the right amplicon are you expecting or not?.

You have a contamination, that could come from a DNA template (genomic) or from an amplicon from a previous PCR. Besides checking the water and other components of the PCR mix, clean the pipettes and the bench where you are working. There are some commercial products to eliminate DNA from surfaces but probably it is enought cleanning with ethanol.

Ideally, have a set of pipettes for preamplification and another for postamplification and separe phisically both steps of the work.

I agree with Rosa, decontaminate all of your work area first (also with UV if available). Include racks, pipettes, cabinet etc. I personally wouldn't bother trying out each component one-by-one, since it may be low-level contamination which doesn't always show up each time you test. I would advise buying in all new reagents to be completely sure (unless you think it could be down to human error i.e. cross contamination by mistake)

Use some RNAase free water and eliquot your primers with this new fresh water

hi, Nisha,

Rosa has given good answer. you have to change the water first, clean you pipettes and also tubes of reagents, this may be contamination from earlier product. or may be aerosol from earlier amplification.

I agree with Rosa and Martin, It is because of contamination u are getting the band in negative control.

most probably the DNA contamination in your pcr reagents or also may be in water. U can try with RNase free water.

for cleaning DNA cleaner is the best option.

@Chris

Yes..this is also a possibility

Hi Nisha

You can clean area of amplification with ethanol , UV . Change your used Deionized water,

maybe your negative control is already contaminated. Is the band size same as your positive control?

Use autoclaved Deionized water to make the fresh aliquot of primers and DNTPs from new stock. Use fresh PCR buffer (new vial) and enzyme. If you are using water as negative control (no DNA in PCR reaction), use autoclaved Deionized water. If you are using genomic DNA as a negative check your primers for false priming.

if you are getting product in desired range of base pair...dont change anything in your master mix. take care regarding annealing temperature and see that there should be not more than 0.5 microl litr of primer per 25 micro litr reaction and also dNTP'S not more than 1.2 micro litr........u go for gradient pcr -1 and +1 degree of annealing temp:) it works all the time:) thank you all the best

Hi dear Nisha

you can strile all the lab equipment for PCR for example filter tips, microtubes and ... . even wash the walls and floor of PCR room and your gown with the bleach water and strile PCR worktable with Ethanole and bleach water, also re-create your buffer, ddH2O, primer stock and use of the new enzyme. I hope your problem solve.

I perfectly agree with Lakshmi Narasimha. You need to do a gradient PCR and check the machine for the cycling conditions, to make sure your program has not been changed or modified by someone else. I have had to struggle with the same problem as mentioned (positive bands in my negative ctrl, which was water), for 6 weeks before noticing the problem my annealing temperature and number of cycles. The annealing temp was reduced by more than 20 oC and the number of cycles increased by 5.

I think then annealing temp is far lower than the Tm of the primers, there's very much unspecific amplification. Hope you find a way through soon.

Good luck as you trouble shoot.

Definitely not Photoshop as that is scientific misconduct and your PI would be very displeased with you. Perform gradient PCR to optimize annealing temperature. Try using DMSO to 5-10%.

Which PCR you are performing? is it RT-PCR?

Accepted  you changed water, buffers, gloves and even the working place .  Still the negative control showing bands? Perform a  PCR without RT enzyme  and/or RNA purification with DNAse digestion incolumn. This will take care of gDNA acting as contamination carried over with RNA. Also you may be dealing with a processed retroviral integration that re-inserted into the host genome  (via a cDNA intermediate and hence you not seeing the introns and PCR amplicon is of same size as your product). 

Design primers spanning across exons to make it fool proof. Use NCBI primer blast to make it happen.

Lots of useful tips to avoid false negative. Thanks to all.