sequencing pcr products

Susanne Rohrer srohrer at immv.unizh.ch
Fri Apr 7 01:33:55 EST 2000



Karen Roberts wrote:

> No one ever sat me down and explained the rationale behind od 260 and
> 280.
> All I know is the conversion factor for ds dna is 50ug/ml and for rna is
> 40ug/ml.  I have also seen a conversion factor of 20ug/ml; what does
> that refer to?

Read Ausubel:Current Protocols in Molecular Biology (The Red Book) Appendix
3D

dsDNA, ssDNA and RNA each have different extinction coefficients at 260nm -
hence the conversion factor. by the way what you remember as 20 may be the
33ug/ml for ssDNA

Protein absorbs at 280 nm (Tyrosine if I remember right). Ratios of 1.8-2
indicate pure DNA.

with RNA I think the Ratios for a good prep are different  but I don't
remember...


> What is the conversion factor to use for quanitating purified pcr
> products?

Same as for dsDNA, I'd think

> Also, when evaluating the quality of a purified pcr product
> (as opposed to say, a "dirty" plamsmid), is there anything that can be
> used as an index of quality like the 260/280 ratio?

If you use a good kit, all contaminants (Nucleotides, oligos) should be
gone.
Watch out for an absorption between 0.2 and 0.8 - around 0.5 is best -
because below 0.2 and above 0.8 your measurements might be wildly
inaccurate (happened to me just 2 days ago - I was off by 50%)

> We are going to use pcr product as our template to sequence.  Is too
> much primer in the sequencing reaction a bad thing?

Yes. It will lead to lots of short extension products and a short read.

good luck

Susanne






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