Hi all,
Recently I keep getting this high molecular weight smear in my PCR, and it is present in negative control.
The smearing is only observed when no amplicon is obtained? Makes me think there must be a contaminant, as PCR has preference or shorter fragment.
I've replaced each reagent systematically to new ones, however the result is still the same.
Are there any suggestion or insight into this problem?
Many thanks for your interest and assistance.
can you clarify the difference between fwd and fwd tag are they different primers and what is the difference please?
Tagged primers have 8 bp sequence at the 5'end that do not match the target template. They should bare little effect on the reaction, 2nd structure formation have been minimised in its design. Purpose of tag sequences are for demultiplexing.
Different priming sequence are labelled on the image as Rev and Rev2
I do not think that I can help here. I think that the smear is not genomic dna as it occurs in the no template blank samples. It may only be a theoretical problem as the tagged primers give excellent results and you may want to increase the annealing temperature either from the start or after a few cycles when the tag has become incorporated into the amplimer as Zhao says . The only (low probability) thing that I can think of is that some bacterial dna remains in the Taq enzyme and you may have a homology to a repeat sequence in the taq dna which can only amplify well when there is no competition from your fungal dna,and then it concatenates with itself to form long smears So in blanks and in inhibited samples you might get this effect but as sample 10 smears this rationale is not convincing but you might want to try a non-taq polymerase from another researcher just in case
G'day,
Check your loading dye. If this becomes contaminated with bacteria or fungi, you can get similar artefacts.
Good luck, AY.
Hi all,
Thank you for all your responses. I will optimise the annealing temperature. The loading dye is new, so there shouldn't be any problem, I'll run a blank lane just in case, perhaps there are obscure chemical interaction happening with the dye and the reaction buffer.
My other concern is the contents of my template will not be of single species. Therefore having the too stringent of a reaction condition may prevent my from capturing as much species as possible.
Hi,
Consider the above given points, as increased ann.tmp lowers non specificity (being seen as faint smear above all of the PCR products loaded).
If the smear in NTC reactions is consistent, you may have to consider these few points,
-contamination around your PCR setup area (use hypochlorite to wipe the every section of your work area but dont incorporate it into your working reagents or tubes, which may spoil reagents and makes them useless)
-primer quality/integrity (i mean slight degradation of your primer molecules, which may lead to amplification / mis amplify any existing contaminating DNA in the reactions including NTC)
-always use 1 / 0.5 X TE to dissolve /make the dilutions of primers, DDI water can cause the acid hydrolysis of primers due to low pH
-Always use aerosol barrier tips (at all the stages of your work to prevent reagent contaminating aerosols)
-at any cost / at any level a no DNA control should not give higher mol weight smears/bands (but they may form self dimers at lower mol weight range in most of the cases below 100bp)
I suspect a minor contamination issue existing with your PCR system, unless this reason, you should not see a smear in No DNA control (but no target controls means, any DNA which doesn't contain target sequence can yield such un predicted high mol weight smears)
Hi Yu-fei Lin
Did you solve the problem and found what those smears were? We are having exactly the same looking smears in our PCR, both no-template controls and some samples. It would be great if you could share your further experience on this issue. Thanx!
@ Miglė Tomkuvienė
Our lab did not resolve the problem in the end. We had suspicions that the smearing were due to contaminants from the environment that acted as PCR inhibitory agents. As the serial dilution of the template slowly improved the smearing but also reduce the intensity of desired band.
When we used pure gDNA from single species extracts the reaction were textbook quality, suggesting the problem is of template origin.
The only thing I could personally suggest is to find ways to further purify your template, check the quality is ok first. Often environmental sample there are huge phenolic/sugar contaminants (A260/A230 readings are usually terrible). Otherwise gel excision is my next go to option.
Hope this helps
We had a similar problem with a simple dilute plasmid-based template containing repeats, and this 1991 paper seemed relevant.
1991 Nucleic Acids Research, Vol. 19, No. 18 5079
"Excessive cycling converts PCR products to random length higher molecular weight fragments"
One of our oligos was fluorescently end-labeled, and the smear clearly contained the label, i.e. they were bona-fide ill-amplified DNA, not a contaminant. Same in this paper (they used Southern blotting).
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