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Title: Can someone please explain how to draw a diagram of a restriction map of a plasmid? Post by: lexus3431 on Sep 14, 2012 The results of gel electrophoresis:EcoRI-70, 30; HaeIII-60, 40;EcoRI + HaeIII-40, 30, 20, 10. I really don't understand how to do this. If anyone could explain I would be eternally grateful.
Title: Can someone please explain how to draw a diagram of a restriction map of a plasmid? Post by: MiiMee on Sep 14, 2012 Ok, I am assuming this question is talking about a hypothetical 100bp plasmid and these fragments are the only ones generated (as opposed to cutting these fragments out of a larger plasmid). I'm ignoring a whole bunch of other scenarios and caveats so as not to confuse things for you!!
Plasmids are circular, so if you get two fragments, that tells you it cuts in two places. Your plasmid has two Eco sites and two Hae sites. If you look at your double digest (Eco+Hae) you see that one of the fragments (40bp) is the same size as one of the Hae fragments. The remaining fragments (30, 20, 10) add up to 60. This tells you that the Eco enzyme has chopped up the 60bp piece into two smaller bits, so both the Eco sites occur within that space. I can't draw a circular structure here, so these diagrams are all linear. Consider a stretch of DNA divided up into 10 x 10bp sections for ease of visualisation. I've put a number at each point: 1-----2-----3-----4-----5----- 6-----7-----8-----9-----10----- Hae gives you 60bp and 40bp, so we can say it cuts at points 1 and 5 (a \ now denotes a cut end) \-----2-----3-----4-----\ \-----6-----7-----8-----9-----10-----\ The 40 bp piece is left alone by Eco, but the 60bp piece is chopped up. There are two possibilities. Eco could cut at points 6 and 9, or at points 7 and 10. It can't be something like 8 and 9 because that would give an Eco-only result of 90 and 10. You get 70 and 30 so you know the Eco sites have to be 30bp apart, somewhere inside the 60bp fragment made by Hae. \-----2-----3-----4-----\ \-----\ \-----7-----8-----\ \-----10-----\ \-----2-----3-----4-----\ \-----6-----\ \-----8-----9-----\ \-----\ If you draw these two possibilities on a circular map, it seems they are the same thing (if you flip one over, the sites line up). However in the real world a plasmid has sequences in it that do things, like genes, promoters, origin of replication, antibiotic resistance, etc. If you imagine there is a gene in the 60bp fragment, knowing whether the first Eco site is 10bp into it or 20bp into it is important. I hope that helps you. It's actually quite difficult to explain properly without being able to draw circular maps! |