What absorbs intensely at 260nm?
(by pow.joshi from gmail.com)
Wed Nov 14 14:13:51 EST 2007
On 14/11/2007, Ed Siefker <ebs15242 from creighton.edu> wrote:
> Pow Joshi wrote:
> > just a quickie: did you use 4microgms of DNA in 4 millilitres of buffer?
> > .... I usually do my ligations in a total of 20 microL volume, so just
> > wondering.
> I'm trying to do an inverse pcr which relies on intra-molecular
> ligations. (see
> http://en.wikipedia.org/wiki/Inverse_polymerase_chain_reaction) So the
> concentration has to be very low in order to fa
> > so the concentration is actually ~13 mg/mL which is'nt so bad, is it?
> Considering there were only 4ug in my entire ligation, there's no way
> that's actually DNA.
yes, of course ..... which is why I was wondering about the concentrations
itself .... it is almost as if you created some DNA :-) ....
So far I know, any system compound with a delocalized electronic system,
can absorb in UV range 260/ 230 and 280 depending on the number of
delocalised double/ triple bonds .... Organic chemists, please feel free to
correct me or give additional information.
> > od 260/280:
> > Double stranded nucleid acid typically has a ratio of 2.... 1.8 is very
> > good.
> Right, a ratio lower than that might indicate some protein
> contamination. A ratio much, much higher than that would indicate what?
> > Methinks you want to check you calculations again:
> > Also, as a precaution, allow the DNA pellet to suspend in the buffer
> > completely....
> > Allow the UV lamp of the spec to warm up for at least 20 min.
> > Blank with the buffer you are diluting the sample with.
> > To verify your spec's fine, check another known sample.
> Yes I follow these best practices. I'm not to worried about this
> experiment, I have more DNA I can religate for inverse pcr. I just like
> to know what happens when things go wrong. I suspect it might be DTT or
> something in the ligase buffer. The large volume I'm ligating in would
> mean that anything that precipitates from the buffer would be present in
> large amounts. Maybe I'll put some DTT in my spec today and see.
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