My PCR products are probably specific (e.g. only 1 band), so usually I use a PCR clean up kit to purify my PCR sample (I only check 2 ul of the PCR product with loading dye on an agarose gel). This is much quicker than extracting the sample completely from agarose. However, by 'accident' I added loading dye to my complete PCR sample. Can I still use a PCR clean up kit to purify my sample? Or does the loading dye interfere in any way?
Yes, you can still use a PCR clean up kit to clean your PCR products. The loading dye shouldn't have any interfere with your PCR products neither with the clean up procedure.
I don't think that the loading dye will interfere the PCR DNA. That is why we mix loading dye with our DNA to run the gel. However, I am not sure whether the eluant will be clean enough for downstream experiments. For example, sequencing and cloning.....Let us know, if you find out.
Another thing is instead of going through a column (kit) to purify it, you can run all your sample (DNA with loading dye) on a gel and do a gel-extraction. Then, for sure every thing will be fine (of course, you will lose some DNA, but you definitely will have some good material for downstream experiments).
Sometimes in gel purification my desired band and dye ran together and I had no problem with cleaning the dye and it worked well for cloning. The dye is washed off in the wash step with buffer containing ethanol.
http://www.real-biotech.com/index.asp?modules=product&files=p1&ID=108
Dear Caro Overmars-Bos,
Loading dye never interferes with DNA run on a gel, you can use it. I suggest you try running your PCR products without purification on gel as control match and see what will appear, the same previous results or not, then you can decide what did happen?
As mentioned earlier, I am not sure that the kit column can remove all the dyes. Some dyes will affect some of your downstream applications:
- See attached document (yellow highlights): " -------- the yellow and blue dyes in the reaction buffer may interfere with these applications. The dyes absorb wavelengths in the range of 225–300nm, making standard A260 readings to determine DNA concentration unreliable. The dyes have excitation peaks at 488nm and in the range of 600–700nm that correspond to the excitation wavelengths commonly used in fluorescence detection instrumentation. For some instrumentation, such as a fluorescent gel scanner that uses a 488nm excitation wavelength, there will be minimal interference since it is the yellow dye that absorbs at this wavelength------.
Yes, It should work perfectly.
You can use PCR cleanup kit without any doubt.
I've done a cleanup on a PCR sample with loading dye many times. It always worked.
Did you have any problem with the sample containing the loading dye? Also, which loading dye did you use?
- Badges
- Science method