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:
- Don’t start a bead cleaning if you are in a hurry
I realised that the beads cleaning is the OPPOSITE of time sensitive. Forget the “mix DNA and SPRI beads and let for 5-15 min at room temperature” idea, it will work but you will lose DNA. If your library or DNA sample is already “borderline”, you are in trouble.
To understand why, think about what is happening when mixing DNA and beads: They have to find each other in a soup of diverse enzymes and other ions. Statistically it takes time for DNA to bind to the beads and the less beads/DNA are available the longer it takes.
My best beads cleaning were when, I would start the beads cleaning, let the DNA and beads bind for 30-90 min (depends on the DNA concentration: the highest, the longest), and then put on the magnet. For the 96 well plates, I would let the DNAs for 30-45 min. For gDNA in eppendorf tubes, I would not put on the magnet before an hour. My best advice is start the SPRI beads cleaning while doing something else and forget about them for a while.
- Mix a lot
The standard protocol says “mix well”. Well, mixing goes with the idea that it helps the DNA and beads to get together.
When doing plate DNA cleaning after digestion-ligation, I would mix by pipetting the beads for about a minut per row. And then let it get together for a “long time”.
Sometimes I noticed bead precipitation. It’s nothing to worry about. The beads that precipitate migrate really easily to the magnet.
- Be sure that all beads move to the magnet area.
Test your patience: Do you think that the liquid is transparent enough, that no beads are in suspension anymore? Move the supernatant to a fresh tube and put on magnet. How many beads from the supernatant go to the magnet? Train your eyes to estimate when it’s really clear enough.
Another trick: Most of the time when the volume is >0.8ml, some beads stay at the surface. Pipet at the bottom to bring these beads closer to the magnet area and let it for a few more minutes. Then remove all supernatant.
- Never let the beads dry
This is especially true for genomic DNA. This will probably make the largest difference. If you let the beads dry, it will be really difficult to release the DNA. For that, remove a maximum of liquid by pipetting and then add immediately your water/tris and resuspend.
For 96 well plates, the same is true. If the beads dry, it will be really difficult to wash them from the side of the well. Resuspending beads can take “forever” and is tedious. To get most of your DNA back, the strategy is: add Tris to all wells first. Then work on resuspending. With the multichannel pipet, it’s a challenging step. Take your time, the beads are in contact with liquid and will not overdry. I also found that the yellow racks are the best for effectively put back the beads into solution. If you look within the well, the contrast between yellow and brown beads will make it possible to see were beads are still on the well.
- A bit more patience..
I have no doubt that by getting to the last step, you want to get these DNA’s out of the tubes/well. Vortex a bit the resuspended beads and give it time to release the DNA. What are a few more minutes of patience at that point?