You can probably use the “freeze-Squeeze” method (no kit required).

1) Excise the band from the gel. Try to exclude as much excess gel as possible.

2) Place gel slice in an eppendorf, place in -20 or preferably -80, for one hour to overnight (colder and/or longer is better..)

3) Place gel slice in an open ended sterile parafilm envelope (pre-prepared)

4) Squeeze the gel slice between your fingers, liquid will come out, collect it using a pipette and transfer to an eppendorf tube.

The quality and yield of DNA may come out a little poor.

What is your downstream application?? mentioning this will be helpful for people to give good advise..

Yield can be increased by very accurate excision of the band from the agarose. If your yield is ok, but concentration is low (as in: you have a couple of hundred uL of squeezed out liquid), then it can be concentrated using ethanol precipitation. Assuming ok yield, you can clean it up and even concentrate it with an appropriate kit (you may have to adjust the elution volume, and be sure that the kit can deal with the small product size!)

Invitrogen claims their kit can elute as small as 40bp, but claims don’t always hold true in one’s own experience.

Many commercial kits don't recover DNA

Is there any other possibility without qiagen kit?

Daniel is right with the size limits of most kits. You can alternatively use an agarase (there are several commercial brands) to digest the agarose gel and then precipitate your fragment. Nevertheless, with such a small fragments you should use some kind of carrier (tRNA, molecular biology grade glycogen...) to facilitate the precipitation

Lots of kit-free methods explained at this RG thread:

https://www.researchgate.net/post/Can_anyone_help_me_to_find_a_protocol_for_gel_extraction_without_using_kits

After melting the gel with the binding buffer, you can add 0.6 volume of Isopropanol. That generally helps in recovery.

If you have to clone your product, you can run a portion of the amplified and precipitate the rest. You should lose a good amount of primers.

I would suggest fermentas gel elution kit. So basically, run the gel and cut it and recover it using the gel elution kit. I would suggest running the PCR products a couple of times and so that you would be able to use it for future use (cloning or sequencing). After the PCR, you can pool the gel pieces and elute it all at once or do it separately and finally after checking it, the eluted products can be pooled. I would also suggest an alternative method which is the ethanol precipitation as suggested by others.

Attached please find an old but trusted protocol that has always worked well in my hands.

You can probably use the “freeze-Squeeze” method (no kit required).

1) Excise the band from the gel. Try to exclude as much excess gel as possible.

2) Place gel slice in an eppendorf, place in -20 or preferably -80, for one hour to overnight (colder and/or longer is better..)

3) Place gel slice in an open ended sterile parafilm envelope (pre-prepared)

4) Squeeze the gel slice between your fingers, liquid will come out, collect it using a pipette and transfer to an eppendorf tube.

The quality and yield of DNA may come out a little poor.

What is your downstream application?? mentioning this will be helpful for people to give good advise..

Yield can be increased by very accurate excision of the band from the agarose. If your yield is ok, but concentration is low (as in: you have a couple of hundred uL of squeezed out liquid), then it can be concentrated using ethanol precipitation. Assuming ok yield, you can clean it up and even concentrate it with an appropriate kit (you may have to adjust the elution volume, and be sure that the kit can deal with the small product size!)

Invitrogen claims their kit can elute as small as 40bp, but claims don’t always hold true in one’s own experience.

Freeze-squeeze should work here, a good version is this protocol: http://personal.bgsu.edu/~gangz/Page099-103_Elution_of_DNA_from_Agarose_Gels.pdf

I used this a lot of times myself with good succes. What I don't know, is how good the recovery rate of small DNA pieces is but adding some Glycogen (10mg should be enough) should help here..

Well I would suggest a couple things.

Since your product is pretty small it is possible that the binding to the invitrogen collumn is not great and you are loosing it in the wash/flowthrough step. We use the Macherey-Nagel PCR purification kit and it works well for a 52 bp fragment. This would be the preferred option

Alternatively, you can run a low melt Agarose gel and then melt the gel and do a phenol-CHCL3 extraction of the DNA (not a great option but it works). You can also run a regular Agarose gel and dissolve it using a Beta Agarase and then extract the PCR fragments

Lastly would be to simply Chloroform extract your PCR reaction, add some Glycogen as a carrier and ethanol precipitate the product

Good luck, Vik

Rather than doing all this,

use Macheryrey-Nagel kit which has the lower limit of eluting upto ~50bp..

Simple

Thank you very much for your valid suggestion, In my future i have to colne that product (insert) into pcDNA3.1+ vector.

Personaly I hate kits.... I like the 'Freeze and squeeze' method described by Nicolai Bonne above. However, agarose and ethidium bromide in my experience give a somewhat 'dirty' product. For a product of this small size I would use a thin non-denaturing TBE acrylamide gel, run a few micrograms of product and mark bands by UV shadowing (no ethidium bromide as this will cary over). Then cut and soak overnight in gel slice vol + 500 ul buffer (anything with more than 200 mM monovalent salt). The next day recover the extra 500 ul and alcohol precipitate. This will give VERY clean product at about 50% yeild.

Gisela Vaitaitis , In our lab -80 c is not working . If i will use -20 overnight or ice cubes for half an hour

Senthil,

Absolutely, you can use -20C overnight! I'm not so sure about ice for 1/2 hour but then again I have never tried that so who knows. Generally it's thought that longer times and freezing conditions are better for precipitation........

Good Luck!

Senthil,

Save yourself and do the -20 over night. This is known to work, I think ice for 30min won't work, as Gisela Vaitaitis explained. The key is to do as cold as possible (-20 and colder is good, don't go warmer) and long enough (over night is guaranteed long enough at -20). You can get away with only 1-2hrs at -80, and you need at least the same or more time when using -20.

"experiments take a lot of time, short cuts make them take longer"

Afrooz Rashnonejad , already i used invitrogen kit, i didnt get products...

Temperature has interestingly a much lower influence on the outcome of the precipitation than usually thought, for details see here: BRL Focus (1985) "Ethanol Precipitation of DNA" http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf/focus.Par.56415.File.dat/Focus%20Volume%207%20Issue%204.PDF

Its important that the mixing is done well and that the salt concentrations fit. If you have only very litte DNA to precipitate use a co-precipitation agent like glycogen.

Thermo Scientific GeneJET Gel Extraction Kit is able to purify fragments from 25 bp long. I use it for my products (approx. 70bp) and it was successful. During the purification I lost around 50% of it but you can not avoid it with such small fragments. Good luck!

I agree with Christian- use a carrier agent for alcohol-based precipitation of low amounts on DNA. Using -80C is a "legacy" method, and is not necessary.

you can use a homemade method of elution of DNA described by Barbieri et al. "Rapid DNA elution procedure from agarose gels." Anal Biochem 235 (1996) 106-107. I use this method and I get high yields.

Vikas Jain, i have ordinary (rankem) isopropanol .. Can i use that one...

Yes, why not? There are no special requirements for molecular biology here, except it shouldn't be contaminated and diluted.

Dear Senthil,

As per the information given by you regarding the pcr amplification, the size of the pcr product is in the range of 70bp which is very small. There is the max possibility of loosing your pcr product during elution because of two possible reasons

1. Check the min and max cut off size for the elution column of invitrogen because while washing product may loose because poor binding to the coloumn

2. Poor precipitation or time of precipitaion.

U can follow the gel extraction and purification procedures given in sambrook. I hope it may works for you. All the best.

if you also have problems with the precipitation of your DNA fragments, I use the precipitation with sodium acetate (3M stock solution, use 1/10 volume of the sample) and absolute ethanol (two volumes respect to the solution with sodium acetate) as shown in Maniatis, for very small DNA fragments. I put O.N. at -20 ° C and centrifugal subsequently to 12000 rpm in centrifuge (MiniSpin) for at least 15 minutes (preferably 20 minutes).

Hi Senthil,

All the above recommendations sound excellent. I can also recommend using the Qiagen Nucleotide Removal Kit for your application:

http://www.qiagen.com/Products/Catalog/Sample-Technologies/DNA-Sample-Technologies/DNA-Cleanup/QIAquick-Nucleotide-Removal-Kit#orderinginformation

I have experience using this Qiagen kit for purifying PCR products as small as 40bp. It's a very quick protocol that can be scaled up to process many samples using a vacuum. Your products look pretty clean from the gel you showed, and as such this kit should be suitable. I believe that a column will give you better yield than using a gel extraction kit, and should be compatible with downstream cloning of your product.

Good luck!

Alex

Yes, Ordinary Isop will work.

Use cut the gel then melt it.. Use conventional ethanol precipitation will work.