PCR Purification: AMPure and Simple
Post date: May 9, 2012 5:16:12 AM
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?