PCR product purification

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douglitas318
douglitas318's picture
PCR product purification

Hello to all, glad to be part of the forums.

I'd like to know how important it is to know the DNA concentration of a given PCR product before purifying it? We are using a kit to purify the PCR product and then using the purified product in an RFLP assay. Several problems with these RFLP assays lead me to suspect that something is wrong with our purification process.

The PI says that finding the DNA concentration in our PCR product (via Qubit) is cost-prohibitive so we just go right to cleaning the product after amplification. Does anyone else go right to cleanup without verifying DNA concentrations, and if so how do you insure that you're not diluting the PCR product too much when eluting in the spin column?

Biju
Biju's picture
Hi Douglitas318

Hi Douglitas318
I will try to answer the questions and clairfy doubts  below.
1. The standard procedure for PCR productpurification  is to run on a gel to ensure PCR amplification of the correct product, purify the product using a spin column technology as used by you, dissolve in the least quantity of solvent(mostly distilled water or a buffer) after elution, quantify using Quibit or spectrophotometer and use for experiments.
2. I do not know anybody who do quantification before the elution is completed.
3. It is important tro use the approprite kit for PCR product purification as the kit selection depends on the product bp size.
4. If water is used for elution, ensure you are using DNAse free water.
5. With experience, you will be able to know the PCR product quantity to be used for RFLP from the gel picture itself avoing the quantifictaion step altogether. 
6. As I mentioned above, elute the product in the minimum volume stated in the kit.
Hope this helps in your intial experiments.
Good luck
Biju Joseph

dav668
dav668's picture
 Hi,

 Hi,
For pcr product purification, first perform pcr to amplify target. after that, perform gel electrophoresis. if u see more than a band, meaning u have to cut gel to obtain specifically ur expected fragment and proceed to purification. after that, quantify ur purified product on gel (semi quantitative) or nanodrop or spectro-. if u saw only an expected band from gel, u can direct proceed to purification followed by quantitation.

pls note some of the kits are dedicated to purify product from gel, some could do pcr product purification directly. some are able to do both.

good luck.

Dav

emmafiori
emmafiori's picture
Hi,

Hi,
I don't bother quantifying my DNA/ PCR product unless it is going to be used for a qPCR standard curve. I just run it on a gel, and if the band is weak, then I start over with a new extraction, or try concentrating the DNA.