I wanted to talk a little about the selection
characteristics of Agencourt’s AMPure beads,
a bead-reagent combination that purifies PCR reactions.
This stuff is incredible in terms of simplicity, efficiency,
and high-throughput compatibility. I have a sneaking suspicion that AMPure, not
unlike fire to Prometheus, was handed down from the gods to benefit humanity. You
just dunk it into your sample, slosh it around, stick it to a magnet, wash,
wash again, and elute in your favorite buffer. No muss, no fuss.
We were wondering, though, about its selection process. What
size fragments are selected by the AMPure beads, specifically at which ratio of
beads to sample? So, like diligent scientists, we rolled up the sleeves of our
labcoats and… read the protocol.
The protocol recommends washing your sample in a 1.8:1 ratio
of beads to sample, although it says that fragments less than 100bp will be
omitted at this ratio, it doesn’t say which sized fragments will be selected.
We found this remarkably helpful technical bulletin, which describes
calibrating each batch of AMPure beads with various ratios of DNA ladder.
So I did our very own calibration with AMPure beads using
Fermentas’s GeneRuler™ Low Range DNA Ladder (25-700 bp). I added 30ul ladder to
various concentrations of AMPure beads according to Agencourt’s instructions.
(Actually, if you’re looking for good AMPure instructions, I
recommend looking at Illumina’s TruSeq™ Sample Preparation Guide. Honestly,
their instructions are more comprehensive than Agencourt’s, and easier to
read.) After purifying each sample, I bookended the various AMPure:ladder
ratios with 10ul non-purified ladder on a 2% TBE gel for easy comparison.
Without any further ado, here are the results:
The results aren’t too surprising, I guess. Unless you’re
looking to select 100-150bp fragments, or if you’re using an extremely low
ratio of AMPure beads, the ratio differences aren’t that significant.
Basically, barring the first exception, you’ll be just fine following
Agencourt’s protocol and recommended ratio.
From this one image, it’s difficult to quantitatively
compare one ratio against another, so I plugged everything into ImageJ to give
me some numbers to play around with. I followed ImageJ’s guidelines for analyzing gel images. Then, I averaged the band intensities for both
non-purified ladder samples, multiplied them by three (knowing that I added
three times more ladder for purification), and normalized the band intensities
of the purified ladder by dividing them by their corresponding band intensities
for the non-purified ladder.
If you didn’t follow the grammatical train wreck that was
the previous sentence, don’t worry, you should just focus on the results:
Interestingly enough, according to ImageJ, the 1.6:1 ratio
has slightly more intense bands, and apparently slightly more purified DNA, than
the recommended 1.8:1 ratio. (If you want to see my exact analysis process, you can view the attached Excel file.) While those
values don’t mean percentages because the normalization isn’t exact, it does
suggest that different AMPure ratios to DNA can produce different results in
terms of fragment size and amount retained.
And, when you really think about it, isn’t that what experimental
PCR purification fragment analysis is all about?