If it is a PCR product you could simply TA clone then digest out fragment from plasmid. Another alternative is to use a PCR clean-up kit without running on gel. Small DNA fragments are not co-purified.

You never quite get total recovery of DNA from DNA clean-up kits so you may just need to digest and run more product. If you still lose product then this might point towards other reasons: make sure you are using the kit correctly.

We used to carry out electroelution followed by ethanol/NaAc ppt etc but the use of kits changed all that and gave much better recovery.

Use the EOH-NaOAc3 precipitation method

Use the EOH-NaOAc3 precipitation method you can find the protocol inthe attachment.

Good luck

Mona

Hi, Soundarya!

You can use magnetic beads.

Ayoob Radhi and Mona Albloushi How do you make sure you precipitate the desired DNA fragment?

Soundarya Priya after precipitation run on the gel only 2µl to check the intensity and the expected size of the fragments.

good luck

Mona

make a primer with biotin label on the 5'end and clean up post digest using streptavidin beads to bind the 5'end fragment and either keep the eluate or discard the eluate and dissociate the biotin labelled fragment from the column depending on which one you want. Alternatively just design a new pcr primer and just amplify the fragment that you want as the only pcr amplimer. Why do you need the digested fragment?

Like Paul, I am guessing that this is a PCR product that you have RE digested. I often had problems with cloning RE digest bands recovered from gels, so now I don't bother, provided that I can heat inactivate the RE. After that I just do an ethanol precipitation (sometimes with glycogen as a co-precipitant), redissolve in ligation buffer, and ligate. I might need to screen a few more clones, but thats not a major hassle.

Alternative option...just switch to Gibbson Assembly cloning, or any of the other homologous end recombination kits like InFusion.

If it is a PCR product you could simply TA clone then digest out fragment from plasmid. Another alternative is to use a PCR clean-up kit without running on gel. Small DNA fragments are not co-purified.

You never quite get total recovery of DNA from DNA clean-up kits so you may just need to digest and run more product. If you still lose product then this might point towards other reasons: make sure you are using the kit correctly.

We used to carry out electroelution followed by ethanol/NaAc ppt etc but the use of kits changed all that and gave much better recovery.

Yes. Using elution from DEAE cellulose paper.

Isolation of DNA from agarose gels using DEAE-paper. Application to ...

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC327265/

by G Winberg - ‎1980 - ‎Cited by 142 - ‎Related articlesJan 25, 1980 - The method involves the simultaneous transfer of all DNA-fragments from an agarose slab gel onto DEAE-cellulose paper and the elution of the ...

Ian: thats a good point, but only if the amplification used taq polymerase. The proof-readers dont add the terminal nucleotides. But one can always do that at the end useing taq.

Margaret: I have searched for DEAE paper many times and found no supplier. If you, or anyone else, can help me find it, I would appreciate that muchly.

You are right Geoff, thanks for the correction, much appreciated. We routinely used Taq post PCR.

If you are trying to size separate restriction fragments, there really is no realistic alternative to gel electrophoresis.

Before you get too worried about A260:280 ratios and recovery rates, do some calculations on what concentrations you are likely to achieve assuming a 50% recovery rate. You are likely to find that the concentrations are below the limit for accurate measurement of A260 and A280. Anything below 0.05 is highly uncertain. Gel electrophoresis is probably a better way of checking recovery. If you include a 2x dilution series of your original digest you can measure recovery by comparison with this. At low concentrations this is more accurate than absorbance measurements.

Hi Soundarya,

As Andrew says , the A260/280 ratios are unreliable for low DNA concentrations , and even when the DNA concentrations are good, the ratios can be pretty awful following gel extraction.

I try to avoid gel extractions where I can, as I'm not very good at them, but I avoid using ultrapure or DEPC treated water (pH of DEPC H2O can be around 3-4) and ultrapure pH 6 ish) water as these both have low pH and, for me, don't elute well from the columns, particularly in small volumes. I use the sterile water from sigma better if I need to avoid using elution buffers.

As for other methods:

If you are digesting a pcr product and the digest leaves only small fragments then most of these wont precipitate well, so just heat inactivate and precipitate as Geoff recommends.

If the other fragments are bigger, do they have the correct ends to go into the multicloning site? If they don't, they might bind one end of the cut vector, but shouldn't circularise.

If they would insert into the vector, do they have a restriction site present that won't be present in the cut vector or the desired insert? If yes, you could add half a ul of this restriction enzyme to the ligation mixture 5 minutes or so before using to transform your competent cells. This will reduce the numbers of the undesirable insert coming through. You can also use this if there is a suitable restriction site in the empty vector.

I haven't worked in research since 1998. I just know that capturing DNA in DEAE paper using electrophoresis is an easy way to isolate fragments from DNA preps. I always did large scale digestions and captured copious amount of my desired fragments.

Margaret: yes indeed. So isnt it strange that you can't get DEAE paper anymore, (while the resin is still available)? Maybe I should invest in a paper maker!

can you use nitrocellulose or nylon and wash the dna off before it sticks permanently @geoff margison ?

Paul; although I never actually tried that, I argued that it wouldnt work. I dont think the interaction with NC/nylon is ionic (but I might be wrong), and eluting with water is going to give SS DNA, even if it works (although a post annealing should cure that problem, of course). I have some bands to purify next week, maybe I'll give it a try! Any thoughts about the buffer for elution?

It would certaily beat electroeluting, which I never really had any sgreat uccess with!

Geoff M,

Maybe I did use the DEAE resin. It has been a long time. I don't remember the substance that I used being paper like. I remember it being membrane like. You could try that as well when you isolate bands next week. Just try it on a band that you are not interested in.

Hi Geoff,

I thought that the first interaction with NC was ionic but the baking bound the DS dna covalently so similar to Southerns the ds dna will stick lightly to the membrane then can be baked on or washed off probably with low ionic strength buffer or water. would love to hear how it works

Margaret: I think it was probably DEAE paper, before the kit era, my colleagies and I (and Paul, I'll wager) used that as a the method of choice (much preferred over low melting point agarose and phenol!).

Paul: did a bit more lit search on that and you are right: people have leached off DNA (well, 18-mer oligos!) from NC using TE with really incredible yields. hope I can fit it in next week, and will let you know outcome.

Apologies to Soundarya for hijacking her question!