I got a single band which represented the actual size for the DNA of interest. I want to purify it before ligating with a cloning vector. Before ligation, I also want to add an A-tail. Which method is the best for DNA purification; excising the desired gel band or purifying the PCR product with a DNA purification kit?
Dear Budak,
Once you received your band, as you said actual size of interest, no need to go from Gel, you can purify your PCR product with any PCR product purification kits, eg: Qiagen. In the case of you didn't get a single band and you got more bands in the gel, you need to excise the gel part, which is your correct one and go for a Gel Purification steps.
Dear Budak,
As Doctor Karuvantevida said, if you have a single band you don't need to excise the gel part. Besides, if you that band is a result of PCR, you already have a A-tail in your amplicons.
Good luck.
Ana
Hi, why you want to add A-TAIL to your PCR product? If you want then clone it in TA vector you can use Taq polymerase . In addition you can use clean up kits for your product as you said it has single band.
@ Sohrab Boozapour: In many cases, such as long amplicons, a need for accuracy and no point mutations, using Taq is generally not recommended. On the flip side, most enzymes which are used for long products and are proofreading do not add A-tails.
@Budak Kuantan: Personally, I have had the most success gel extracting the band, adding the A-tail afterwards in new buffer, dATP, Mg, and Taq than adding the A tail in the original PCR product mix. I suspect it's a combination of non-optimal buffers, exhausted dATP, competition of the original polymerase, and the fact that it's not a pure solution of product.
I have noticed in difficult to topo-clone/clone in general inserts, that a simple PCR purification will still leave impurities of different sizes which is enough to throw off your downstream reactions. However, in most applications, a PCR purification when you have a dominant single band would be sufficient. It's also cleaner in terms of carry over contaminants from the actual gel extraction, so I would recommend trying a pcr purification at first.
Cheers and good luck!
Yes you have to cut the band and purify it from the agarose using a commercial kit. Very easy. It takes only 10 minutes.
If you have used a plasmid to obtain your fragment (e.g. by PCR) you need to purify it from an agarose gel, especially if you need to clone it into another plasmid.
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