NAPS, the UBC service we use for Sanger sequencing, charges 2 CAD for purifying a sequencing reaction; especially if submitting several reactions, there is therefore a reasonably big incentive in doing the purification ourselves. Continue reading
SPRI beads cleaning is one of the most repetitively used step during library preparations and probably the step where most of us lose a lot of precious DNAs.
Losing DNA scared me so much (because it can be observable) that I hesitated a lot before trying to use beads to concentrate genomic DNA, because usual rate of recovery are ~50%.
Hopefully with some practice and a lot of patience, it is possible to reach 90% recovery. How to lose as little DNA as possible? Here are some guidelines: Continue reading
A few weeks ago Kristin posted a nice blog entry on home made SPRI beads, which effectively replace the ridiculously expensive commercial AMPure beads. (Editor’s note: see this post at RLR for more about the AMPure beads). Dan already ordered the ingredients to make them in our lab as well, and when they all arrived I volunteered to prepare and test them. Following are some results. If you don’t feel like reading through the whole post, the bottom line is that they work very well, and that you are very welcome to use them (they are in an aluminium-wrapped Falcon tube in the fridge, with a white “SPRI beads” label).
AMPure XP beads are used to quickly clean PCR reactions and are also used in WGS library prep. You can also use them to do DNA size selections! They work well, but are very expensive. I found step-by-step instructions on how to make a home-made version online, purchased the reagents and tried the protocol. I tested the beads by attempting to clean up DNA ladder and I found that the home-made beads work identically to the AMPure XP beads.
Attached is the protocol for making the beads. I followed the protocol exactly. One note, I weighed the PEG-8000 in the fume hood because the fine powder is an irritant if inhaled. I also have attached a picture of my gel comparing clean-up of 100 bp DNA ladder with commercial AMPure XP beads and the home-made substitute.
Ampure XP magnetic beads have become popular for cleaning DNA from reactions and for size selecting DNA. WGS sequencing library preparation protocols, and the GBS protocol, involve one or more bead clean up steps. If you make next gen sequencing libraries you will use Ampure XP beads. Following is some basic information about them and my thoughts about using them, mainly for library preparations . . . Continue reading