hyper supercoid band

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shpungin
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hyper supercoid band

Post by shpungin » Fri Jul 14, 2006 6:48 am

some time when I separate cut and uncut vector with one site only, I can see that the supercoil and relaxed form are now all liniar at the right size , but there is a weak band which is smaller than the supercoil. I call it hypersupecoil. this band is also in the uncut vector. it seems that in this form of plasmid the digestion is inhibited.

does anyone has this problem also? do you have an explanation?

last time I purified KS on biorad quantum maxi prep and I got quit large amount of this hyper supercoil which I couldnt digest.

can it be due to proteins bound to the DNA ?problem with the purification proccess?

too many bacteria on one column, which didnt allow geting rid of all protein bound to DNA?

herb386
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Post by herb386 » Fri Jul 14, 2006 10:16 am

Sounds strange. Have you found this result more than once? Perhaps your cells have two populations of plasmids, one of which is either a completely different plasmid or one of them has a mutation at the cut site. You could try sequencing DNA from the "hyper supercoiled" band using primers flanking the cut site. If you get a band with the right sequence then I have no idea. If you get no band then it's probably a contamination in your sample. Either another plasmid or a DNA fragment cut from left over genomic DNA.

I usually find there is a small amount of genomic DNA left over whenever I extract plasmids so the most likely explaination is that your digestion is cutting a fragment out of this. Do you purify the plasmid using a gel after extracing it from the cells?

You do get different levels of supercoiling of plasmids within bacteria so it may be that the most supercoiled ones can't be cut. Or as you say it may be due to bound proteins.

shpungin
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Post by shpungin » Sat Jul 15, 2006 8:02 am

It might be a contamination,but since I have 3 mg of this preparation I didnt want to throw it away, and i dont want to clean from gel every time i will need it.

incase that it is over load of germ in the preparation maybe I can do phenol chlorofom extraction to get read of all proteins.

the point is that it does happen when i do mini preps , there is this hyperform. and when i run on gel cut and uncut i can see it in both samples. i allways ignore it and i keep on going with the cloning and it s ok and not contamination.

this why i wonder if the possibility of very tight form cover with protein ans hard to digest does exist?

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