PCR product and Restriction products bands are not of the expected size - (Oct/29/2014 )

Hello, I am performing PCR and restriction for VDR using Taq I. I have gotten PCR bands that are not of the expected size (740 bp) first image {IMG_0269e} I get around 500 bp according to Fermentas Ladder fourth image {sm038_fam}

Also after digestion the bands are still not of the expected sizes:

TT-> 245 and 495

Tt-> 495, 205, 245, and 290

tt-> 205, 245, and 290

Images two and three {IMG_0243e} and {IMG_0282_2}

P.S. For the restriction I put in a tube: 3.5 ul PCR products, 1ul provided buffer, 0.5 Taq I restriction enzyme, and 5 ul nuclease-free water (I use New England Biolabs CutSmart restriction enzyme with its buffer)

I incubate for 35 minutes at 65 oC then run them in 2% gel

Well, the gels and digestions look fine. The problem is likely the choice of primer binding locations.

You can make your gel look more uniform by adding some dye to the positive electrode (I assume you are adding dye to your agarose). This will keep the dye from migrating to the negative electrode, which causes the shadowing you see.

phage434 on Wed Oct 29 12:18:10 2014 said:

Well, the gels and digestions look fine. The problem is likely the choice of primer binding locations.

You can make your gel look more uniform by adding some dye to the positive electrode (I assume you are adding dye to your agarose). This will keep the dye from migrating to the negative electrode, which causes the shadowing you see.

 Thank you for answering.

First; my primer is the same with Riggs et al. Here is a link for the article that used the same primer set (http://www.apocpcontrol.org/paper_file/issue_abs/Volume6_No2/Hemant%20K%20Bid.pdf)

Second; I add Ethidium Bromide while melting the agarose and add loading dye for samples; should I add Ethidium Bromide to the buffer in the chamber to remove the shadowing?

Yes, you can add ethidium bromide to the buffer at the positive electrode to prevent this shadowing.  Of course, it would also help to run your gels longer.

phage434 on Thu Oct 30 00:33:39 2014 said:

Yes, you can add ethidium bromide to the buffer at the positive electrode to prevent this shadowing.  Of course, it would also help to run your gels longer.

Thank you for that point; I will try it the next time I go to the lab, but do you have any suggestion regarding my PCR band that comes out 500 bp instead of the expected 740 bp? It's affecting my results a great deal