Gel Shift Question
genecutl at mendel.berkeley.edu
Tue Mar 8 07:47:43 EST 1994
In article <smrodems-080394082728 at f181-208.net.wisc.edu>,
smrodems at students.wisc.edu (Steve Rodems) wrote:
> I have started to do gel shift experiments and I have a question about
> doing competition studies. My probe is about 30bp. Now, in a competition
> study I use cold 30bp DNA as a specific competitor and linear BS/KS+ as
> non-specific competitor (ie, should not compete off my specific complex).
> If I want 100-fold molar xs of competitor I can add 100ng cold 30bp DNA per
> 1 ng 30bp probe. My question is how much BS do I use? 100-fold molar xs
> of BS actually has about 100x as many 30-bp sites since each molecule of BS
> has about 100 30bp sites (that would actually be a 10,000 molar xs of 30-bp
> sites if I'm thinking about this right). So, what is the conventional way
> to do this, molar xs of a whole piece of DNA or molar xs of available x-bp
> binding sites?
I think the best calculate the amounts of nonspecific competitor to use
is to go by moles of nucleotide rather than moles of plasmid. For example,
1 mole of 30bp oligo = 30 moles of base pairs, 1 mole of 5kb plasmid =
5000 moles of base pairs.
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