Different Patterns on Different Agaroses.... - ...Why? (May/20/2008 )
I run the same PCR products from the same PCR reaction mix on different agaroses (Metaphor from Lonza and Agarose 1000 from Invitrogen), all other conditions the same, and I get similar yet slightly different patterns of bands. Please see attached photos for an example.
Why???
Those are not very different.
I acknowlege that they are fairly similar but there are still bands on the agarose that don't appear to be there on Metaphor. When one band different = different type assignment then that is a fairly big deal.
I acknowlege that they are fairly similar but there are still bands on the agarose that don't appear to be there on Metaphor. When one band different = different type assignment then that is a fairly big deal.
Actually I have no idea (which size are the bands?). But if you use for all experiments the agarose of one supplier it should be no problem.
Metaphor agarose is use when you have small fragments or when need a higher resolution. Some times I combine metaphor with another agarose 1:1. If you have fragments less than 150bp or several fragments with a few bp of difference between them use metaphor. If fragments are bigger or the difference between them are more than 75bp use a regular molecular grade agarose. Preparing metaphor is a hassle so use only if really needed.
200-600bp in size.
I always use the same agarose but another lab uses metaphor and we want to be able to compare results.
It's less a practical question and more a simple curiosity question.
Which is exactly why I originally decided not to use it even though the other lab did.
Which is exactly why I originally decided not to use it even though the other lab did.
If you used for both the same solution, perhaps a mix problem in the pcr mix , or problems with gel homogeneity? EtBr distribution changes?
Only guesses...
Only guesses...
Thanks for the guesses but I know it's none of those. It is a consistently repeatable phenomenon and the only difference is agarose type.
My guess is differences in the agarose (for example different impurities for each type), will lead to slight differences in the cross linking during gelification, which results in differences in the pores size and therefore a minor, yet noticeable, change in the pattern of the bands.
Not sure is that of course, just an idea.