keep raising the annealing temperature until either the other band disappears or the pcr fails up to 70C. If this does not work then try adding DMSO at 2. 4. 6. 8% at a working temperature. Go back to 1.5mM Mg for these experiments as more MG makes pcr less specific

Was your template gDNA? Your non-specific band looks really strong, not as if its simply spurious priming. It could be either alternative splicing, or amplification off gDNA and mRNA. Good luck!

Hello Hiasindh,

what is purpose of your experiment? Some downstream analysis as a sequencing of PCR product? If yes, you can easily cut out the desired electrophoresis band and clean up using some commercial column kit (http://www.mn-net.com/Products/DNAandRNApurification/Cleanup/NucleoSpinGelandPCRCleanup/tabid/1452/language/en-US/Default.aspx) or following this cost-free protocol:

A Quick, Cost-Free Method of Purification of DNA Fragments from Agarose Gel

Hope this help

keep raising the annealing temperature until either the other band disappears or the pcr fails up to 70C. If this does not work then try adding DMSO at 2. 4. 6. 8% at a working temperature. Go back to 1.5mM Mg for these experiments as more MG makes pcr less specific

I agree with Martin Bartas. you can cut out your desired band from the gel. Run the gel slowly to separate out the bands so that it is easier to cut the desired band. You can use gel elution kits to recover your band from the gel. Also do not use the large comb to make he wells in agarose gel since the bands are very close its wont be easy to separate. Instead load on the same type of wells as shown in picture. Good Luck

Thanks to all for your suggestions.

To Craig Silver,

The template is parasite's genomic DNA only, cultured strain in laboratory. Pure culture.

To Martin Bartas and Sheetal Bandhu,

We are planning to use this PCR for detection of drug resistant in clinical samples. So, its not just for downstream processing. Its needs to be implied in field clinical samples. So I have to remove the non-specificity.

To Paul Rutland,

I am going to perform PCR by increasing the annealing temp, as suggested. I hope i get my desired result. Will update the result tomorrow.

Thank you once again to all.

Hi Hiasindh,

What are you PCR cycling conditions?

In fig. 2 you have a much weaker secondary band in lane 8, you could repeat this as before but reduce the Ta length to 10 - 15 second.

The longer the Ta, the greater the amount of non-specific annealing.

Of course you can cut the band as suggested, sequence to verify and use the purified product as DNA template for further PCR amplification. If you do this, use a high fidelity polymerase (Pfu, Phusion or my personal favourite - Q5), there are plenty to chose from.

I'm excited to see how it goes,

Best wishes,

Daniel

Hi Daniel Brady,

PCR conditions are:

1. Initial denaturation - 95 °C for 5 min

2. Denaturation - 94 °C for 1 min

3. Annealing - for 45 sec (trying gradient)

4. Elongation - 72 °C for 45 sec

Step 2-4 for 30 cycles

5. Final elongation - 72 °C for 7 min

The PCR product length is 701 bp, so I have kept 45 sec for annealing.

Thanks for your inputs.

Try designing a new set of primers for the same gene, primer locations away from the first ones, so that amplified fragment still covers the required region. Best of luck.

You can use touch down PCR cycle with starting annealing temperature as high 68 or 69 C depending upon which temperature you got the best bands. If for e.g, its 58 C, use 68 C in the first cycle and then gradually bring annealing temp down to 58 C (1 C) in each cycle and then rest of the 25 or 30 cycles on 58 C.

You can also try using Ammonium sulphate PCR Buffer to reduce non-specificity if it really is a non-specific band.

Hi Hiasindh Ashmi

For me I Had this before in one way I checked the GC content for gene in interest if it is high I add 5% DMSO to PCR reaction. And in other way iI just cut out the my a certain band, gel purification and used it as templet again.

good luck,

Hi, Hiasindh Ashmi

you can try reducing the concentration of gDNA (a DNA curve would be an option) and using the minimal concentration of MgCl2 for all samples in the curve

Based on the brightness of both bands, I think you'll just have to design new primers. Is your gene of interest a single copy in the genome or is it part of a gene family? You might have to sequence both of your PCR products to find a region to use to make specific primers.

Hi,

As suggested above, increasing Mg will only reduce the stringency...

You may also change the Polymerase being employed for PCR amplification, In certain caseswe have observed such doublets and they disappear when a polymerase is changed. In an old article from NAR, it was shown that looping out of template due to hair-pin formation between repetitive region is resulting into some lower sized (than expected) bands. The probelm was resolved by adding SSB protein in the PCR, The SSB keeps the DNA in SS form, which resulted in amplification of correct sized product. See if these suggestions can resolve the issue

bye

please change polymerase to high-fidelity one.

partially digest template with non-cutter

They are both spesific bands. Getting an extra band does not always mean non-spesifity. Your primers probably bind and extend 2 different loci.

Please blast your primers via NCBI tool. you will probably get 2 match.

Ashmi,

Until you have sequenced both bands its hard to determine if the "non-specific" band isn't an actual product from the genome you are working with. I concur with previous researchers that you may want to redesign the primers or optimize your PCR reaction further. However, sequencing both bands (Sanger sequencing is relatively cheap nowadays and there are numerous commercial providers) and blasting the bands against NCBI would be the more methodical approach, and will ultimately help you focus your efforts more effectively.

Good luck.

Hi

After PCR, what is your downstream process?

If its for cloning, you could elute the band of interest, further resolving the gel, as there is 100bp difference between the specific & non-specific one.

I would recommend you to cut your desired band and then do gel pruirification. you can also putify your PCR product, by a PCR purification kit. Then try to increase the annealing temperature as a parellel strategy. use positive controls to check the PCR kit quality. For example, use a PCR product as a template and see if your master mix will amplifiy it as one clear band or not. If it applified it as one clear band then the annealing temperature is the problem if you obtained two bands then the master mix is the problem.

i would suggest you to add 3% DMSO and to increase the annealing Temperature. It would work.

Please check different conditions:

Try different kits for DNA isolation

Try ready-made lyophilised Master Mix for direct PCR amplification

Try different PCR conditions and also PCR machines

Use specified kits for gel elution and plasmid purification

Hi Hiasindh Ashmi

You should cut your 700bp band and do downstreaming part.Sometym its happens when you use Hf enzyme.

As others said, reduce the Mg to a minimum. Probably your buffer already has some Mg, so do not add any more. Do a temperature gradient using this conditions. To be clear: more Mg reduces the specificity

Indeed, increasing Mg-concentration will give more non- specific products. The explanation is that positively charged Mg2+ will form salt bridges between negatively charged phosphate groups and stabilise all primer-binding (increase the Tm); so more miss-primed products will be generated. I don't think its a problem with the enzyme.

It can well be that the primer concentration is too high. I advice to perform a chessboard titration from 32 nmol primer up to, say, 512 nmol per reaction and lower you Mg2+ in 2X steps. Choose the best combination.

It is also a good advice to sequence your products. Cut them from the gel, clean them up and get them sequenced.

However, even if you primers seem to bee correct; redesign with a good primer program such as AllelID might be the cheapest choice. I used primer 3 and Allel ID next to each other; Allel ID outperformde primer 3 to a great extent in my research questions

I would try different primer pairs first .

I would set up test with several groups, with 5-6 samples and controls in each group. The only difference between group is annealing temperature. You may find a best annealing temperature.

Hi, the bands are really strong. I once had that problem but my non-specific amplifications were not that very strong. So I did a lot of PCR optimization; reducing the MgCl conc., increasing the annealing temperature, reduction in primer conc. But the most successful was increasing the annealing temperature. But in this case the band is too strong and so like the other authors said, the best thing to do is just to cut out your required band from the gel and purify. Qiagen have good gel purification mini kits for this.

Hi dear,

Once I had faced the same problem. I played around with the annealing temperature and it started working.

All the Best!

Most of the opinions are interestingly good to try with, playing with higher annealing temp above 60 or changing MgCl2 conc from 1.5-4.0 or even adding PCR additives as DMSO or Betaine in higher G-C ratio (not in case of Pfcrt gene).

But it is important to know whether the other band (500-600bp) are really non-specific! You didn't include a positive control in your PCR run. Then you need to cut both of the band's and sequencing them to know their nature whether there are a part of malaria DNA or of human DNA nature. (P. falciparum 3D7 strain is to be used as a positive control).

Second, what is the purpose of your amplification? Mostly , those researchers who are targeting Pfcrt gene (1275bp exons length) are going to do either gene sequencing or RFLP-PCR for detecting gene mutations at 72 until 371 codons. So if you are going to do, you need to amplify at least 900bp (without primers length) to cover the whole mutation area, not a 700bp.

On the other hand, it will be very difficult to find suitable REs for RFLP at that length using your designed primer pair; the longer PCR product, the higher number of RE cut motifs, and accordingly difficulty in interpretations.

This article might be useful:

"Djimde at al., 2001. A molecular marker for chloroquine-resistant falciparum malaria. N Eng J Med, 344: 257-263"

Thank you so much.

Hi Hiasindh Ashmi ,

I think so stranger to have a non-specific band pretty close of the correct fragment. Did you consider a possibility of a second (but specific) amplification? Might have a deletion? For response this, I think which you can cut the bands and perform a sequencing for each fragment (if the in silico analysis is so specific for your primers).

Regards,

Hi Hiasindh Ashmi ,

Here are my recommendations:

1. Add 3%DMSO or 2.5M Betaine to the PCR Cocktail.

2. increase the annealing temperature upto 70C, but this might bring down the intensity of the desired product

3.Primer redesign may be the next step if the issue persists.

4. Lastly you might elute the non-specific band from gel and purify and sequence it to confirm its origin.

Hope it helps!

Hi everyone,

Thank you for your support.

I have redesigned the primers and it is working perfectly.

Thank you once again to everyone.

Hi Hiasindh

What is the Tm of your both new primers?

and what is the annealing temperature of your PCR reaction?

Best way is to add 3% DMSO and check it with gradient PCR with annealing temperature increased 1C upto 5C from your previous Tm. It always work

You can cut only a specific band.

Increase annealing temperature up to 68. Even if it does not disappear please try boiling the PCR product at 100 degree for 5 minutes followed by ice chilling for 10 minutes. Hopefully it will work

Dhirendra Kumar Can you tell me annealing temperature ? I already tried so many temperature but did not get band ....

You can try touch down temperature in PCR.