Why can’t I use a spectrophotometer to measure my mini-prep DNA?
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 spectrophotometer! 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proliferation of those and other small-volume specs in modern molecular biology labs has made this discussion somewhat out-of-date.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! Use our Spec Reading Calculator to see if your miniprep quantitations are reasonable.
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 the 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. On a Nanodrop spec, make sure you clean the pedestal frequently, and periodically reblank the device, otherwise the readings can be quite incorrect.
- 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!