One of the most common problems I
encounter as a Sequencing Core Director is that
Core clients often try to measure the concentration of their
mini-prepped plasmids or PCR products
using a spectrophotometer. Some of you may be able to do so, but most of you will
not get reliable readings. There
is not enough DNA in the typical mini-prep or PCR
to register on the typical spectrophotometers found in most labs. Most
of the time,
you'll be reading dust or air bubbles or simply baseline drift. You
need to be very
cautious with your results - unless you have a spec with a cuvette volume of
10 ul (or perhaps as high as 100 ul, depending on the specifics of your
sample).
Here's an example calculation based on a
mini-prep.
If your sample is
a PCR reaction or a gel-eluted fragment, read this section, then see
additional comments
at bottom.
Consider the following:
- Let's say you are growing a 1 ml culture of pUC19. Plasmids
usually yield between 1 and 5 ug per ml of culture. For pUC19, the
yield is often at the high end. For argument's sake, let's say your
yield is on the high end - 5 ug.
- After the prep, you resuspend your DNA in, say, 50 ul and
transfer a 5 ul aliquot into your cuvette that contains 0.5 ml of ddw.
What kind of A-260 can you expect? About 0.02 AU.
- MOST SPECS ARE NOT AT ALL ACCURATE AT 0.02 AU.
- In the above scenario, you might be able to get a
reading by dissolving your sample in 20 ul instead of 50. Now you might
expect a reading of 0.05 AU.
- Many specs can read at 0.05, but only if they are
very carefully blanked.
- Your spec reading will be worsened by any of the following:
- Dissolving the prep in more like 50 ul and trying to
read 0.02 AU. No way.
- Adding just 1 or 2 ul to your cuvette and trying to
read 0.01 or 0.02 AU. No way.
- Getting a reading of 0.14 and not being
suspicious. There's no way a 1 ml miniprep will give you 12 ug of DNA.
Usually you have RNA or chromosomal DNA instead. This is the number
one cause of failed sequencing with minipreps.
- Your spec may be able to take readings on less that
0.5 ml, but make VERY sure you have the cuvette aligned correctly so
you aren't reading absorbance through the meniscus or through air
bubbles trapped in the cuvette.
- Let's face it - mini-preps don't often yield 5 ug of
quality plasmid from each ml of culture. If you've got an expression
plasmid or a cross-species shuttle vector or some other low-yield
plasmid, don't expect anywhere near that amount. Be glad if you see 1
ug from a ml of culture. Be suspicious if it's more.
- Do you blank your spec both before and
after the reading, just to be sure? You should, if you're trying to get
good readings around 0.05.
Here's the bottom line - you should
be very suspicious of a spectrophotometric reading on mini-preps,
PCR reactions or gel-eluted fragments. Yes, it's possible, especially
if:
- you have a high yield plasmid
- you have a spec that can read small volumes (<100
ul)
- you have carefully blanked the machine, preferably
both before and after taking the reading
- you are realistic about how much DNA you expected vs.
your spec reading.
|
We recommend running a mini-gel on your DNA as a check of
concentration. Cut your circular plasmids to linearize them, and run
alongside a DNA of known concentration that's about
the same size.
How about PCR reactions or
gel-eluted fragments?
You'll see the same problem - PCR and gel elution often do not produce
enough DNA
to reliably measure spectrophotometrically. A PCR reaction of 20 ul
usually gives
on the order of 1 ug of product, which can be measured only if you have
a spec that
can read sample sizes more like 10 ul. A gel elution may start
with a lot of
plasmid DNA, but after you cut and gel elute, there's often precious
little left. Be
suspicious!
|
