PreSPRI pooling

[jm googlecalendar]

In gearing up for our first try at Smart-3Seq for LCM FFPE sections the protocol (page 14) specifies a second cleaning by resuspending in 102 uL of water/SPRI breed mix (60/42 uL = 0.7 bead ratio).   This is straightforward.

I am, however, trying to reconcile that with the Option 2 protocol on page 5, which specifies a bead mix of 36 uL of "SPRI bead mix or bead binding buffer.  I understand the standard protocol uses a bead ratio of 0.6, however, I am uncertain exactly the use of this buffer here?  If this is a control ("or") I am unsure what it might be controlling for at this point in the protocol? (Typically, we would use "no-template" or "no-RT" as a control.

I didn't pick this up until going through the (incredibly useful) spreadsheet of consumables/hardware/etc. for ordering purposes, which lists the bead binding buffer.

Please advise and many thanks,

John Matyas, PhD

Professor, Faculty of Veterinary Medicine

University of Calgary

[jm googlecalendar]

My apologies for not clicking previously on the link on how to make SPRI beads in my garage...  I find your very clearly described recipe for bead mix:

https://openwetware.org/wiki/SPRI_bead_mix#Beads

And, an equally clearly written protocol for buffer:

https://openwetware.org/wiki/SPRI_bead_binding_buffer

And the sentence that I am pretty sure explains the use of buffer at the stage cited above:

"After the DNA/RNA is purified on the beads, they can be resuspended in a reaction mix and left in during the reaction ("with-bead protocol"); afterward, this buffer will rebind them to the same beads, saving on the cost of bead mix (it is equivalent to the buffer used in the bead mix)."

I take this mean that the bead binding buffer is the _full substitute_ for another dose of "fresh beads".  Oui?

j

[Joe Foley]

Yes, in the second SPRI step of that version of the Smart-3SEQ protocol you can replace the complete bead mix with just the beadless buffer, to reuse the beads that are still in your tube from the first SPRI and save some money.

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[jm googlecalendar]

Sweet.  Thanks.

j