I would expect 45 bp to separate from primers  very well on a 3% low gelling agarose but if it were my problem I would Amplify ( assuming this is a pcr product) then add exonuclease1 to the mixture which will destroy all single stranded primer making column purification better as there will be no primer sticking to the column matrix. Then gel separation or, if the product is clean, just column purify the product. If it is not pcr product then we need to know the sizes of any other fragments that need purifying away

Thanks Paul for your reply. Actually I am PCR amplifying a library of 125 bp. then digesting it with restriction enzyme. It will make fall outs of 80 bp and 45 bp. I need to use the 45 bp for further cloning process. While purifying very small fragments (e.g 45 bp) from gel by column we loose a lot of DNA. Therefore I was wondering if there are any other method by which we can increase the yield. 

ok here are 2 threads from previous researchers..

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

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

some points: 1  minimise uv exposure when viewing the gel.

The freeze and squeeze technique where you cut the gel slice and freeze it and spin out the dna works well.

If all else fails there is a technigue where you cut the gel in front of the band you want and insert a slice of deae cellulose  membrane .continue electrophoresis and the dna sticks to the membrane. View the gel again and if the band has vanished remove the membrane and elute off the dna. A variant is to cut a well in front of the band,continue electrophoresis and pipette out the buffer in the wel and measure the dna directly) but these are rather dated methods now.

If you are using columns bind the dna for longer than recommended and elute twice with a longer on column elution step.

Not mentioned is cutting the band out and digesting the agarose away with agarase.

When precipitating the dna as the yield and size are small add glycogen  to bulk up the precipitate when ethanol precipitating the dna . Glycogen is a polysaccharide so should not interfere with downstream applications or quantification

good luck,

Paul

Just as an alternative, Anion Exchange and Ion-Pair Liquid Chromatography is another method you can use for separating and purifying DNA. 

cut and separate the desired band with scalpel blade then purify would get pure and clean DNA

cutting out band is one option. look around for an electroelution apparatus. if yes, run labeled oligos for start and finish.

Pauls' idea of DEAE paper is fine, but I have searched high and low for DEAE paper (to do just what he suggests) and it seems that no-one makes it any more! Please supply a link if this is not the case.

And relating to another thread, we had suggested hitting the unwanted fragment with one or more RE then use the resulting mix for cloning  without any separation. Not sure if this is feasible in your case of using a "125bp library", although you do cut with one RE, so presumably you know that that site is present? But then, why not use a different pair of primers that generates just what you need?

Geoff, Annealing two primers for creating a RE site is not possible in this case because we do not know the exact sequence since I am dealing with a library.

Well I had tested 3 different methods to purify 45bp fragment after running the agarose gel and cutting the band from gel. 1) Gene jet gel extraction kit from thermoscientific. 2) freeze and squeeze method 3) electro-elusion method. Last two methods were followed by precipitation with Glycogen (10ug/ul) and absolute ethanol (keeping at -80 overnight). At the end we ran PAGE gel to check the DNA concentration by staining with syber gold. The recovery of DNA was best in Electro-elusion method. Thanks everyone for your advices.

Good that something worked. But I am having problems understanding what you have done (and why). So you amplified your library: presumably you used two primers for that (rather than primer extension)? Then you digest to get 80 and 45 bp fragments. So you must know that the RE site is present, so it might be that that site was in one of the primers? If so, that is a mighty big primer to use!  More likely, perhaps is that the RE site is in a non-variable region of the library?  Its all  very intriguing: please can you clarify?

Hi Geoff,

You are right that GE, Whatman and Camlab all seem to have discontinued de81 papers so my advice was rather dated on that method. It is possible that hybondN+ or Hybond xl will work ( and maybe a small amount behind your sample to stop other dna from catching up if it does work)

Hi Geoff,

In the pass I have made DNA-fragments untill 45 bp long (single strand) and we purify them with an anion-exhange like MonoQ  under basic conditions (the G base also deprotonate and give even separation between DNA-fragments with the same lengh and different bases). Dionex (now Thermo Fisher) has also a very good HPLC-column for it named  DNApac PA100. After purification with a gradient of NaCl or KCl (pH=12 with NH4OH) you can easily desalt your product with a G25 DNA-grade gelfiltration column.

Hi,

i have purified 30 bp ds DNA fragment by  gel extraction procedure using qiagen products. Yield will be around 50% compared to that of longer length products but thats enough for cloning purpose.  you can digest more amount of template to get good amount of digested product.

Hi Geoff, You can find in this file for your clarification.

Thanks Paul. It is true that we can not buy the same membrane once it is over. Next time I will try with Hybond to see if it works.

Thanks for the diagram: now its clear!

Are these REs the same or different? In either case, I don't think you need to purify the RE digest product before ligation. If the sites are the same, then I would carefully dephosphorylate the vector and check that by self ligation. I would then consider a range of insert:vector molar ratios (starting at 1:1 and moving towards 1:10) in attempts to obtain plasmids that have only one insert. And I would do this also if the sites are different.

I wonder if orientation is going to be critical, and how you would plan to assess this?

Hi Geoff, Exactly! I needed to purify the RE digestion product (45bp) from gel very efficiently, therefore I asked question! :-)  I did experiments and saw eletro-elution is working best. We are cutting with one RE (BsaI) which cuts DNA unevenly therefore we do not need to dephosphorylate or worry about the orientation.