DNA conc by EtBr staining?
bawagan at umdnj.edu
Wed Jan 12 13:15:13 EST 1994
He said it. We used what Mr. Rasmussen had said.
Rasmussen at Bioscience.utah.edu (Randy P. Rasmussen) writes:
>In article <2gkfpv$kb2 at mserv1.dl.ac.uk>, jclewley at crc.ac.uk (Dr. J.P.
>> 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
>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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