Why Can't I use a Spectrophotometer to check my Mini-Preps (or PCR products or gel-eluted fragments)?
This should be subtitled 'your tech told me there wasn't enough DNA in my sample, but I quantitated it with a spectrophotomter! It MUST be right!'
|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).
Note: The Nanodrop UV Spec is an example of a system that is capable of making reliable measurements on minipreps. The proliferation of those and other small-volume specs in modern molecular biology labs has made this discussion somewhat out-of-date. Still many of the points I make below are still valid, even if you use a Nanodrop!
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:
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!