What absorbs intensely at 260nm?

Ed Siefker via methods%40net.bio.net (by ebs15242 from creighton.edu)
Wed Nov 14 11:17:09 EST 2007


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.


> 
> 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.
-Ed



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