|Apr17-12, 06:10 AM||#1|
DNA agarose gel: one than one chromosome, one band.
If I run an agarose gel of undigested DNA of an organism with more than one chromosome, and obtain one band (slightly smeared), what does this mean? The chromosomes aren't all the same length (some might be approximately half the size of others). Is it because they all become "entangled?", they run a distance shorter (i.e. suggesting they are larger) than any one chromosome. One could even speculate that the band has migrated a distance expected for the genome size, but this might be influenced by the above thought (since the resolution at that large a size makes it difficult to tell).
Any help appreciated,
|Apr17-12, 10:44 AM||#2|
Aha! I found pulse field gel electrophoresis, and on wiki it says the following: "DNA molecules larger than 15-20kb migrating through a gel will essentially move together in a size-independent manner."
So my undigested DNA moves at the same rate, apparently. EDIT: realised a part doesn't make sense so it is removed: I misinterpreted what it meant and in also in a way ignored what it meant.
This makes sense for why an organism with more than one chromosome shows one band. However, with a small insertion, the migration through the gell is retarded, this is not size-independent, then. If they move indpendent of size then an insertion would make no difference, so there would be no difference in migration between large DNA with or without an insertion. My results would make more sense if the DNA became 'entangled', that way insertions can be additive, which would account for a smalll inseertion making a large difference in migration, and at the same time explain (maybe) why there is only one band for an organism with multiple chromosomes.
Has any one heard of multiple chromosomes becoming entangled?
Thanks in advance.
|Apr18-12, 08:11 AM||#3|
Basically chromosomes are just too big to be effectively separated with conventional gel electrophoresis, you need pulsed field gel electrophoresis, or possibly liquid chromatography with a really really long column length. Depending on how big the difference between chromosomes in basepairs is, you may need quite a long time to detect the differences in length (i.e. 100bp difference between chromosomes will take much longer to resolve than a difference of 20kbp)
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