What absorbs intensely at 260nm?

Ed Siefker via methods%40net.bio.net (by ebs15242 from creighton.edu)
Tue Nov 13 12:37:39 EST 2007


I got some really strange readings on my spec.  For the inverse
PCR I'm planning to do I ligated 4ug of genomic DNA in a volume
of 4 ml with NEB's T4 ligase and buffer.  After ligation I added
400ul of NaAc and 9ml EtOH.  I precipitated it, washed with 70%
EtOH, air dried, and resuspended in 10mM Tris.

On checking the concentration with a 1:25 dilution the readings
were off the scale.  I tried again with a 1:100 dilution the A260
was still extremely high it was at least readable. The concentration
it gave me was 13685ng/ul and the ratios 260/230 and 260/280 were
both over 3.  Adding more buffer pushed those ratios up to 6.
Something is clearly very wrong here.  I checked some .5ug/ul lambda
hindiii ladder and it read ok.  What could have possibly gotten in
my DNA to do this?




More information about the Methods mailing list