I would start by decreasing the amount of template by a factor of 10 at least. Pcr works on copy number so if you consider 20ng of human genomic dna with a size of 3billion bases and 2 copies of the target amplifies ok think how many copies of your target there are in 10ng of a genome size maybe 8kb.

I think if you cut out the correct band and column purify it will be fine

Decrease the elongation time to 30 sec, which would get rid of the un-specific band and can be used directly for cloning. Also, if you decrease the number of cycles (e. g., if you are using 35 cycles, now go for 25 cycles), the un-specific band will not get amplified.

Good luck

You can refer to a similar question:

https://www.researchgate.net/post/How-to-reduce-or-eliminate-the-non-specific-amplification-in-PCR

I agree with Shirin Parizad that a reduction of elongation time is really helpful. From my experience, a reduction of this time by factor four helped to avoid unspecific bands (from 20 sec to 5 sec in case of a 144 bp PCR-product).

When you need this PCR product for cloning, you could perform a gel extraction of this band, since your band has a relatively wide distance to the unspecific band.

I hope this helps a little bit :)

Thanks for the answers.

I used 1ng template instead of 10 and 25 cycles instead of 35. The elongation time was already 30 sec, so I didn't change it. The non-specific bands didn't go away, but they did get fainter.

The fact is I'm going to perform restriction-digestion cloning with these products, and since I don't have access to a gel extraction kit, I'd have to do PCR clean up.

Do you think these results are good enough for that?

Is there anything else I could do to get a better outcome?

Would it be a good idea if I did PCR clean up and amplified the extracted DNA afterwards?

I'd really appreciate your help.

OK since less template has not cleaned the pcr if all that you are interested in is the restriction digest I would run more cycles to get a strong signal and just cut the dirty product. Check if your enzymes cuts well in pcr buffer. New england biolabs publish this information. Your wrong band will cut the same in all samples and is quite weak so the stronger amplimer cut products should be readable. Remember that as fragments get smaller they take up less dye and look fainter.

If you really want to clean up the product without using pcr cleanup kits you could try a reamplification with nested primers or even just one nested primer which often gets rid of non specific amplification but I would cut the crude amplimer that you already have

@ Paul Rutland sorry if I didn't explain my situation very well. I do have a pcr clean up kit, but I don't have gel extraction kit.

A pcr cleanup kit will clean both amplimers so does not solve your problem. If you are worried about the larger band you could try running the product, dipping a plastic tip into the good nand ,dip the tip into a new pcr mix and re amplify. What is the next step in your experimentation....it may have an effect on advice if we knew what is happening and why Paul Weber but at the moment it still looks like a workable plan to digest the impure amplimer

@Paul Weber I've send you an answer which deals with the mixture of your "own" gel extraction kits. In this answer, a recipe is described how you can perform gel extraction without kit.

Maybe this could be the easier way than optimizing the PCR.

@ Paul Rutland the next step is to cut the PCR product with two restriction enzymes, insert it into another plasmid, and use it as an expression vector.

In that case I would cut the crude product and clone it since the minor product is so weak but if you want a simple and cheap gel purification then freeze and squeeze is quick and easy. Cut the gel band out ,freeze it 3 or 4 times with thawing in between and remove the liquid which will contain your amplimer. Losses are high but you do not need much product to clone Paul Weber

If you want to try this low percentages of agarose give a better yield than high concentrations

Thank you very much.

Just a quick question. To be honest, I've never done DNA gel extraction. If I did purify from the gel, would I get enough DNA concentration?

I'm asking because we usually use 300-400ng in a 20µl rxn for restriction digestion.

Hi Paul Weber , may I ask what polymerase you are using? The normal polymerase has a processivity of 1kb/min and a good efficient polymerase can do 1kb in 30 sec. That being said if 30sec didn't get rid off the longer band then you can decrease the elongation time to 20 sec.

Sayanta Bera thanks for the comment. I'm using a Taq polymerase, and the same reagents that I have always used. Now that you mentioned the elongation time, I also do an extra elongation step for 5min at 72C in the end. Do you think It'd be better if I wouldn't do that final step?

On the basis that you can just see about 5ng of dna in agarose with ETBr I would say that your original strong samples could easily be 600 ng so even if you lost half in freeze/thaw you should have half retrievable after spinning down in the supernatant

Paul Rutland thanks a lot. Can I ask which method would you choose: using a gel extraction kit which expired 1-2 years ago (kept in good conditions and never opened) or the freeze and squeeze method?

The reagents and columns in the kits are very stable but losses are quite high on purification but I would expect the kit to work. I would try freeze/thaw and assess the yield ( blank the nanodrop with TBE if that was your running buffer). It is the quicker option and try the kit if yields are unacceptable

You can still do the last step of 5 min elongation. The critical elongation step is during the cycles. If 20 seconds is enough to exclude the long fragment's amplification then one last step of 5 min elongation should not matter.

Thanks for all the answers.

Decreasing the elongation time to 20, 10, and 5sec didn't work.

Eventually, I cut the correct band and used the expired kit to extract the PCR product.

I loaded 40ul of the product and got around 3.5ug of DNA back in an overall volume of 35ul ddH2O.

Use gel extraction kit and elite the pcr product.

Paul Weber Which kit did you use? Can you share the catalogue number/company please?