Are you running a water no template dna control and does this NTC amplify. If so you have pcr contamination. If not then possibly your primers are annealing to some common sequence in the genome and need further redesigning.

Also try pcr with one primer only. It should fail of course but if you have a viral sequence primer they often anneal all over the place and find another primer extending from the other direction giving bands.

Your annealing temperature does seem very low for oligos with Tm greater than 59, It may be worth checking the sequence that it is annealing to

If you are designing new primers make sure that at least one of the primers is outside of the ones that you have used before then previous contamination cannot amplify

Is it possible that all of your samples are,in fact, wild type and the experiment has not been successful?

What is the difference in result for the WT and the mutated samples.....a size difference or failure to amplify of one type

After seeing your 1st answer I have blast both primers in NCBI nucleotide BLAST and I have found that Forward primers having 100% similar sequence with Rice genome (Chrosome4) Not the RP. Is it this possible that this is an reason that I have getting bands in all samples? No in both WT and my mutated samples I have getting same length of bands 281bp, there is no difference.

The problem of getting band in every samples is continued. This time I have designed new pair of primer sets, One primer set is on flanking region of gRNA on T-DNA and another primer set I have designed on the Cas9 region on T-DNA. Before ordering this primers I have blast both the primer sets and those are not binding in any genomic DNA region of any organism. Then when it came ,For dilution of Primer mother stock I have used fresh autoclaved water from other lab and pipettes also I have used from another lab.

Before doing PCR I have Isolated fresh genomic DNA and all reagents I have prepared in fresh. Then I have put PCR but again I have got bands in every sample including Wild type and Water control also. after this experiment I thought primer might contaminated or PCR pre-mix has contaminated,

so next PCR I did on another lab and only new aliquot of Primer and new isolated genomic DNA, and this time one thing I also Included that I have used genomic DNA of Chickpea, Medicago and Peanut with two water control but again I got bands in all of my 5 samples with 2 rice wild type , out of 2water control one had very very faint band other not and in chickpea and peanut I got band not in Medicago.

The primer I had used

FP Primer Tm 64

RP primer Tm 65

I used annealing temperature 60, and amplicon size is 580bp

So, now What I will do?

For more clarification of my problem our lab have used more than 10 pairs of primer and all the time we are getting same result. Frequently one thing happen in our lab is that primers are getting contaminated but sometimes the primers are ok and we didn't get any band in wild type and water control but band in every samples then we go for sequencing with or without cloning in PJET vector, then also we noticed that the gRNA which should be present on that clone that gRNA is not mine that gRNA from my another lab mates cloned gRNA, that clone He or she still didn't use for regenerating plants, It means the contamination of his or her cloned vector in my PCR reaction but in which stage this contamination is coming we can't understand -

1) During isolation of genomic DNA?

2) during PCR?

3) During PCR elution?

or 3) During sequencing?

or 4) those all primers no are not good for genotyping?

Now What should I do?

Dear Ashis

PCR contamination is a huge problem and extremely difficult to clean up. It may be necessary to clean the entire laboratory and all equipment or move the work to another laboratory. A colleague in a commercial lab told me that it took $64000 to clean up his laboratory of pcr contamination.

Autoclaving will not destroy dna….it sterilises water but dna can still remain in the water but the results that you report sound more like dust contamianation with old pcr product. So the dust is in the circulating air or attached to duct which may get disturbed and contaminate a reagent.

You mention that the primers do not BLAST but be careful with Blast. Its default setting is to exclude repeat and viral sequences from the search because they are so common and will report too many hits so check the settings of your Blast search.

Often the only way to deal with contamination is to amplify a larger product. So if the contamination amplimer is 600 bases then any amplimer of 650 bases can only amplify from genomic dna and not from the old pcr product.Is it possible to clone your product into another vector which has sequences different from pJET so that you can design primers that amplify from the new vector but not work on the old dna just in case the contamination is plasmid with insert not pcr product.

You ask where the contamination is entering your system. We are talking tiny amounts of dna. Remember that 10 cycles of pcr is 1000 times more product and 20 cycles is 1000000 times more product. The contamination could be spread on duct particles and get into the dna preparation by gravity or in centrifugation steps.Preparing new dna in another lab might clarify if it is in the dna preparation but the important stage is the pcr which amplifies the contamination very efficiently and the best way to stop this is to design new primers so that the pcr cannot amplify the contamination. The problem is not in the sequencing or the dna elution steps .It is being amplified and that is the problem

Best wishes,

Paul

Thank you so much sir, I will do my whole experiment from DNA isolation to PCR by using everything from another lab and lets see what I will get. I will update the result after few days.

Dear sir(Paul),

I am very happy to tell you that our PCR contamination problem has resolved. As you suggested that most probably it was dust contamination after read my discussion and also you suggested that if possible we should shift entire laboratory to another lab or we can do our work on another laboratory. As per your second suggestion we ordered all new chemicals for genotyping also the old primer but newly ordered also all the things like tips, MCTs , Tip Boxes etc. And then I had started genotyping on that new lab and I have got very good result in every time, no bands in wild type and water control, also band was in only the positive plants not in all samples. Then I have eluted the PCR product and gave for sequencing there also It was only single peak not multiple peak in gRNA region.

Thank you so much sir, I am grateful to have you as such a excellent advisor. Our entire institute never thought the contamination will be in dust. I learned lot of things from this experiment. And lot of thanks to you to response my every query and gave your valuable advise.

It was a huge achievement from our lab because our lab was suffering from more than one year and you are the one and only spotlight of our darkest path. Our whole lab members with my supervisor(Dr. Hasthi Ram, Scientist III, NIPGR) are very much thankful for you.

I have an request to you, I always want to connect with you so if you don't mind can I get your mail id, I can directly connect with you.

With regards

Ashis majhi

(Your student)