Crude Plasmid Digests - Help! (Oct/01/2005 )
i have a question for my molecular biology tutorial that i dont understand, and i am wondering if anyone here can help me.
I'll write out the whole question...i have some of the answer, i'll write it in caps, would you also be able to tell me if you think its right? thatnks so much!!!
In crude preparations of small plasmids, as many as 2 to 4 plasmid bands can be seen in an agarose gel profile
(1) identify/name all 4 plasmid band typrs from lowest to highest position on the gel
SUPERCOILED MONOMER, RELAXED MONOMER, SUPERCOILED DIMER, RELAXED DIMER
(2)when this multi-plasmid sample is digested with a restriction enzyme, and the products are run in an agarose gelm only a single band is seen. Explain this result with a diagram.
I really appreciate your help all!!!
it is not really a 'multi-plasmid' sample...it is all one plasmid, but the DNA will exist in varying forms.
to understand supercoiled vs relaxed, my old mentor explained it like this:
do you remember those old phone cords, that would get all tangled around themselves and knot up in a big coiled mess? this is analogous to supercoiled DNA, which exists in this form frequently to save space inside the cell. relaxed means there's a little nick in the DNA on one side of the backbone so it won't coil up like that, the steric pressure is released. the cell will nick it's DNA on purpose when it needs to access a strand (during replication, transcription, etc) but most of your DNA exists in the supercoiled form most of the time (the DNA from one of your cells is around 2 meters long if you stretched it all out; supercoiling packs it in there very tightly)
in a solution on the bench, plasmid DNA is floating around in the buffer and will exist in multiple forms, but it's all the same plasmid. so, when you cut it (complete digestion, of course) you only see one band on the gel because all of the DNA will exist in the same form, that of linear DNA.
Does anyone else have anything to add? I think I remember everything correctly but that class was a long time ago
I don't know about the dimer form -- seems to me those are kinda rare...
The three major forms of undigested plasmid, as I recall, are supercoiled circular, relaxed (aka nicked) circular, and linear (mostly due to breakage during purification, I'd guess). Add to this some minor dimer form, and you have four?
I would like to thank snobunny for having put up this particular question. I have my answers for which thanks to every respondent. But I also would like to know at what percentage of agarose gel would it be appropriate to run a pUC18 vector? Actually the vector is one recovered by alkaline lysis method from transformant.
It depends on the insert you have cloned in the vector. If we´re talking about pUC18 alone, 1% agarose works fine (pUC is about 3 kb).
We use 0.7% gels for routine, day to day stuff. It separates well in the 2 to 5 kb range...
And make sure that you use TAE buffer the resolution for the highest molecular weight is better than TBE buffer which I favoured for running pCR fragments under 1Kb
pesji
Yes -- a good point, pesji -- we do use TAE.
Well done Home Brew
I realize that many students don't really know what is the difference between both buffer systems
TAE better resolution for large size DNA (>1Kb to < 30Kb) but heats up pretty fast if you run too fast
TBE better resolution for small size DNA(>50bp to < 3Kb) but does not heats up pretty fast if you run fast (I did up to 180 volts)
They use to be a tab in the Maniatis to show the resolution of both buffers
pesji