Smeary band on low melting point agarose-Why?

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Pleiadesgirl
Pleiadesgirl's picture
Smeary band on low melting point agarose-Why?

I am trying to gel extract my digested large-ish (8 kb) vector using low melting point agarose. Since its large, I have been using 0.8% to try to get good separation, but every gel I've been running is a smeary mess! I thought that it may be due to overloading of DNA, so I've played with that, and have ruled it out. I'm wondering whether it may be due to the low % of the low melting point agarose--I'm new to this type of agarose, so I don't know if this is a factor. Any ideas? I'm running the gels at 90V for about an hour, so I don't think this is an issue either.

Jason King
Jason King's picture
LMP gels take longer to set

LMP gels take longer to set than standard agarose gels. In the summer (without air-con) they don't set. If the gel had not set then you might have noticed that the wells were not as well defined as usual. There could have been bits of aragose lurking at the bottom of the wells. Bands run on such a gel will not be very crisp.

Another thing that could cause what you describe is a buffer mis-match (agarose made up with water and run in TAE or TBE could look like this).

For cloning bits of plasmid DNA I now only use normal agarose. Are you sure you need LMP agarose?