DNA conc by EtBr staining?

Randy P. Rasmussen Rasmussen at Bioscience.utah.edu
Mon Jan 10 13:30:08 EST 1994


In article <2gkfpv$kb2 at mserv1.dl.ac.uk>, jclewley at crc.ac.uk (Dr. J.P.
Clewley) wrote:

> We have been estimating the amount of DNA present in PCR products
> prior to sequencing by comparison with markers, and have got what we
> think are under-estimates.
>  
>  
> We run 1 ug of BRL 1 kb ladder on a 1% agarose gel and estimate the
> amount of PCR product by comparison with the 1600 bp band, which
> we calculate to be about 20 ng. If we then use the recommended amount
> for the ABI dye terminator cycle sequencing kit based on this, the gel is
> overloaded.
>  
Using the 1 kb ladder is probably not the best approach.  I once called BRL
about these makers and they told me that while the 1600 bp band contained
about 10% of the total DNA, this proportion is not consistent from batch to
batch and they did not recommend using it for quantitation.  Try using the
lambda-hindIII digest, the different sizes of fragments give different
amounts of DNA and so you have a "standard curve" of sorts.  If you put 1
microgram of lambda-hindIII on a gel the 23 kb band will have 480 ngs of
DNA, the 9.4 kb band will have 190 ngs, the 6.6 kb band will have 140 ngs,
the 4.4 kb band will have 90 ngs, the 2.3 kb band will have 48 ngs, the 2.0
band will have 42 ngs, the 500 bp band will have 12 ngs ( you probably
would not see this band).  If you need smaller makers then you can use
other digests and do the same kind of analysis - the HaeIII digest of
PhiX174 for instance gives bands between 1.4 kb and 70 bps. 



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