Hello Ahmet

You make a PCR for a fragment, which you want to clone. Depending on your polymerase you will have blunt ends (with proof reading, recommended for cloning) or with 3'-dA ends (w/o proof reading, e.g. Taq). The PCR-product is confirmed by agarose electrophoresis and purified. I suppose that with the primers you introduced a restriction site for sticky end cloning. After cutting (which may be not very efficient when at the very end) now you are purifying your cut PCR-product by agarose gel electrophoresis. Right?

Well, in this case I would recommend:

1) Use a commercial PCR-product purification kit (adsorb your PCR-product at a silica column, clean it from small fragments that do not adsorb) and elute with TE.

2) Quick and dirty would be an alcoholic precipitation (-20 °C over night, add glycogen). Very small fragments usually do not precipitate. Next day centrifuge your fragment, discharge the supernatant, evaporate EtOH and resuspend in TE.

I hope it helps

Kind regards

Peter

As suggested above, I would recommend that you use a PCR purification kit instead of the gel extraction, since you will get rid of the tiny fragments that you get after the digestion of your PCR product. Anyway, it depends on the agarose percentage that you use that the yield may vary in gel extraction, or the size of your product.

Hope it helps!

avid

Hi there!

I wanted to know few things before I suggest anything. 

1. Do you find this band even after your PCR or it just appears after gel purification? Kindly load at each step to pinpoint where this higher molecular weight band appears?

2. If you have purified your PCR product after electrophoresis then I'm sure you would have excluded any other bands! Then after elution and before stting up the digestion did you run the eluted DNA on the gel to check its quality? If the band appears only then, there is something to do with your elution procedure.

3. If the band doesn't appear even then, and only appears after the digestion (when you want to elute the digested insert), then it has got to do with your digestion. 

4. What is the size of your amplicon  and what percentage of gel do you use?

During gel purification using kits, we use the binding buffer (which contains chaotropic reagents like guanidine HCL, guanidine thiocyanate, urea etc.to dissolve the agarose) and heat the gel pieces. This process at times causes denaturation of DNA in an irreversible manner. So the heat denatured or sheared DNA which you get after elution might appear as more than one band of your desired length. Due to random shearing, the sizes will be different than your amplicon. I used to get a very faint and higher molecular weight band after elution but it never interferend with my cloning efficiency. Moreover, the columns provided in gel purification kits are meant to capture smaller and linearized DNA. It won't be able to purify so high molecular weight DNA that won't come out of the gel.

To avoid using the harsh treatment, before you add the dissolved gel to column, let it cool slowly, allowing it some time to renature before proceeding with it. So, even if there is partial denaturation, it might be able to renature back and then elute it carefully.

As for improving the yield of gel purification you can go through the link. I generally use a) 0.6% gel (lesser agarose boud to your DNA, easy to melt), b) incubate for a minute or so in the column every time, c) give multiple washes, d) dry the column to remove ethanol, e) heat the elution buffer to get best results.

Another suggestion is if you are getting a more or less specific amplicon then use a PCR clean up kit where you don't have to heat your DNA and rather than going for purification from gel.

Hope it helps!

http://bitesizebio.com/13506/10-tips-for-better-dna-gel-extraction-results/

http://bitesizebio.com/13508/5-more-tips-for-dna-gel-extraction/

Thank you all very much,

Dear Peter,

Column purification after digestion may not be helpful. Because it will collect all DNA longer than 50bp, which will include many DNA that cut by restriction enzymes. I don't know threshold for EtOH precipitation but I guess it won't be precise either. That is why I need to use gel purification.

Dear David,

I will consider agarose percentage again but it may not be biggest issue because I didn't have a problem with that so far.

Dear Tias,

There is band (2.7kb) after PCR (attached). I did EtOH purification but didn't run a gel after that, I will run it. But, previously I had this same problem and it happens during digestion.

I always use 1% gel without any problem. But I'll reduce agarose percentage. Using elution buffer could increase yield but because I'll do ligation, I prefer using water to elute DNA.

Thank you very much for your responses

If you're going to be cloning this product, why does the low yield matter? You should still be able to do the cloning even with a poor gel extraction. I'd continue forward with the ligation and transformation and see if it works. Only go back and troubleshoot if you fail to subclone.

http://bitesizebio.com/13508/5-more-tips-for-dna-gel-extraction/

Hi there!

You can use the heated elution buffer and use the elute for ligation. I have always used elution buffer and that has not affected my getting positive clones at all. Rather elution buffer is TE which would prevent any kind of nuclease activity but water makes your insert susceptible to nucleolytic cleavage. About gel percentage, I used to run in 0.8% but using 0.6% gives me almost 90% elution efficiency. You'll have ample elutes insert to set up as many ligations as you want.

You still have not told your insert size so it is hard to comment but if it is close to 2.7 Kb then it might be an altered digestion product. Since it only appears after digestion, either your insert contains more than one site for the restriction enzyme you are using. You can digest your vector with those enzymes and check if you still get this higher molecular weight band or not. Then you would know, one of your reagent is contaminated. Set some other digestion using same stock of reagents with same enzymes and look for this band. 

follow this http://bitesizebio.com/13506/10-tip...-better-dna-gel-extraction-results/

it may help you