Dear Colleague,

Gel-purification, when you get such a nice clear DNA band, could maybe not be necessary!

What we do in my lab is that, when the PCR product gives an intense band onto the agaraose gel, we simply directly digest the PCR solution adding the restriction enzyme to the PCR mixture and supplementing with NaCl if necessary (old school manner I agree). Then, after a 1-hour incubation, we quench the reaction by a phenol-chloroform-isoamylacohol treatment, and we directly use the aqueous phase as such for cloning. Sometimes, we take the time to precipitate by ethanol-acetate treatment the DNA in order to get rid of some unknown chemicals present in the aqueous phase after phenol-choloroform treatment that are inhibitory for cloning process.

In fact, we try as much as possible to not use so-called "gel extraction " kits.

Another method used, that gives splendid results most of the time, is to use a special agarose that melt at 37°C. You run an agarose gel (which was prepared in the cold room) at .8% of this special melting agarose (NuSieve for instance would be quite OK for teh size of your amplicon), we color the gel, cut the agarose surrounding the band of the desired size and ... at the moment to digest or clone the PCR product, we put the mlcrotube containing teh cut agaorse band at 37°C, it melts and the you can pipet up 5 microliters of the melted agarose and do what you have to do with this agrose being a solution. It works quite well.

Best regards and good luck for your experiment,

Prof. P. Urban

Yes in general, a "gel purification kit" will lose most of your DNA.  There are ideal cases where the yield is high, and that's of course the figure they're putting into the manual.  But in most real cases, if your DNA deviates in any small way from the ideal model, you can be assured to lose almost everything.  And that's universal, with every vendor I've tried, which included quite a few. 

That said, I have successfully cloned inserts from as little as 5 ng/ul, if it's highly pure.  So you can give it a shot, see what happens. 

If that doesn't do it for you, or if it's not pure enough, check out my blog for some alternative manual methods, which can be a lot more successful (I'm not trying to sell you anything here -- just trying to make you not buy the unnecessary and counterproductive "gel prep kits" :).  http://www.geneandcell.com/blogs/molecular-biology-methods/10883773-dna-purification-from-agarose-gels.  

Dear Colleague,

Gel-purification, when you get such a nice clear DNA band, could maybe not be necessary!

What we do in my lab is that, when the PCR product gives an intense band onto the agaraose gel, we simply directly digest the PCR solution adding the restriction enzyme to the PCR mixture and supplementing with NaCl if necessary (old school manner I agree). Then, after a 1-hour incubation, we quench the reaction by a phenol-chloroform-isoamylacohol treatment, and we directly use the aqueous phase as such for cloning. Sometimes, we take the time to precipitate by ethanol-acetate treatment the DNA in order to get rid of some unknown chemicals present in the aqueous phase after phenol-choloroform treatment that are inhibitory for cloning process.

In fact, we try as much as possible to not use so-called "gel extraction " kits.

Another method used, that gives splendid results most of the time, is to use a special agarose that melt at 37°C. You run an agarose gel (which was prepared in the cold room) at .8% of this special melting agarose (NuSieve for instance would be quite OK for teh size of your amplicon), we color the gel, cut the agarose surrounding the band of the desired size and ... at the moment to digest or clone the PCR product, we put the mlcrotube containing teh cut agaorse band at 37°C, it melts and the you can pipet up 5 microliters of the melted agarose and do what you have to do with this agrose being a solution. It works quite well.

Best regards and good luck for your experiment,

Prof. P. Urban

Hi,

another method to "extract" your DNA from the Agarose Gel is a pretty simple one: Just incubate the gel slice over night in a suitable buffer (I normally used a simple PBS one) and the lowest amount of liquid necessary to completely cover the gel slice. The DNA will migrate from the gel slice into the aqueous solution and you are good to go.

Worked most of the time when the product is pure enough.

Hope it works for you.

Greetings 

In my experience, nanodrop machines are notoriously inaccurate, however the concentration of post-purified PCR products isn't as much a concern for me as getting the correct molar ratio of insert to vector.  Most of my ligations are done in 10uL or less, and sometimes with concentrations much less than what you have.  44ng per uL is ample insert to clone with.  I would simply proceed with your restriction digestions, heat inactivate, and perform your ligations/transformations and see what happens.  You can always adjust the insert:vector ratios if you need to.  

To answer your question about loss, yes as others have mentioned, gel-purification often reduces yield.  You can always opt to run a small sample of your PCR and if it's clean and there are no non-specific bands, you can even go right from your PCR product to restriction digestion/ligations without "purifying". In fact, many enzymes work wonderfully in PCR buffers (I can't remember the paper that looked at commonly used restriction enzymes and their effectiveness in standard PCR buffers but you'd be surprised at how many RE's cut just fine in PCR buffer).  

Thanks to all for sharing your thoughts. Your suggestions helped me a lot. I will proceed to cloning, hoping for good results. Thanks to everyone.

According to me better to purify the PCR products either with column based PCR purification kits or through Phenol: chloroform purification. In many cases as many suggested if the PCR product is clean enough you can directly go for Restriction digestion but it may possible that your digested fragments may cause problems during ligation as PCR mixture contain some residual DNA Polymerase which have the 3' exo activity like Pwo etc which will chew your restriction digested fragments.

All the best

Dear All,

Another recipee from the "old pot" back at a time in which no kit was existing !! Yes I'm 'that' old :) I am among those who introduced PCR in my country in 1988.

So ... dear Abdullah, you can simply run a regular agarose gel, cut the right DNA band and put it as such in a microtube without any added liquid. Put the microtube at -20°C overnight. The day after, take out the microtube at room temerature and wait your DNA slice thaws. When it's so, take the DNA slice out of the microtube and put it inside a Parafilm sheet first folded and put the DNA slice inside the Parafilm sheet right at the fold. Then press the DNA agarose slice through the Parafilm between your tomb and your index finger and take up with a pipetman the liquid released that way on both side of the DNA slice right in the fold. Do that 3-4 times until no more liquid can be taken from agarose that, meanwhile, became very dry.

Most of your DNA is in the liquid. No need of any kit indeed! But after that, you will need to do a phenol-chloroform before proceeding further.

Best,

Salaam

Philippe

Thank you very much Sir Philippe Urban. Your comments and suggestions are very useful. Highly obliged. Thanks

Hello Abdullah,

Normally I use the PCR kit extraction (Qiagen) with good results. Off-course you will lose some DNA but not all. What I do is to decrease the volume of the elution buffer (water) from 50ul to 30-35ul. In average what I get is between 40-60ng/ul and its more than enough to cloning. Once I got clones using 15ng/ul of my purified insert (1.7Kb). My suggestion, go straight with the PCR extraction if it´s necessary it will not take longer than 15minutes. Good luck!

I must say I really enjoyed reading Dr. Urban's answers. Thank you so much for those recipes from the old pot! I have always been quite skeptical of these 'kits'. Thank you once again for the suggestions!! 

P.S. : That idea of digestion in PCR mix is really superb! Saves so much of time!!!