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Old 07-07-2016, 09:45 PM   #1
SunPenguin
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Default ss cDNA SPRI vs regular SPRI protocol?

I'm looking to add a SPRI cleaning step into my workflow after the cDNA generation. Right now, I use the Clontech Smartscribe enzyme to generate my cDNA from RNA. I usually just use some portion of the reaction into a 50uL reaction volume for amplification.

I've heard that some people have had success using Ampure beads to clean the cDNA, then either using the elute, or directly amplify by adding the beads to PCR reaction.

I'm wondering is there a different proportion/protocol for using these beads on single strand products compared to double strand products (as they usually are in regular PCR)? I know the old clontech protocol seems to use SPRI beads to clean cDNA, but the new ones don't seem to bother.
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Old 07-08-2016, 08:51 AM   #2
cmbetts
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You can treat it just like dsDNA. I've noticed that AmpureXP beads consistently have lower recovery on ssDNA, but work very well for mRNA/DNA hybrids like first strand cDNA.
Why would you want to add a cleanup to the reaction, though? It reduces sensitivity while also increasing protocol time and cost. The older SMARTer protocols generally called for only using a fraction of the cDNA downstream because the previous PCR enzyme was inhibited by the RT components, but with the newer, more robust enzymes you can just dump in the whole RT reaction.
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Old 07-08-2016, 09:41 AM   #3
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Well, the trouble is that I had already adopted the protocol to amplified genes of interests using an older enzyme (Phusion). I tried other enzymes (SeqAmp that clontech uses, Q5, and to a lesser extent Kapa), but they just don't work as well.

What kind of RT components would be inhibiting, other than excess KCl and MgCl2? DTT should be inert (I actually dial it down a bit in the RT anyway), and the Tris-HCl seems to be at the same pH as most polymerases.

I also do worry about what all the excess TSO is doing in the PCR reaction... Nobody seems to have trouble with it, but I would think those TSO can also act as regular primers? unless the rG's/+G's (LNA) would inhibit that? but couldn't the polymerase just chew back on the mismatches?
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Old 07-08-2016, 11:05 AM   #4
cmbetts
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I'm not quite sure what causes the inhibition, but I've seen quite a bit of literature about excess RT inhibiting particularly qPCR, but also some single cell workflows (The Quartz-Seq paper showed that the RT was inhibiting ExTaq and got around it using MightyAmp).
That makes sense as a reason to add the cleanup. I'd recommend eluting the cDNA from the beads before amplification. In my previous position, I screened several polymerases for an on-bead amplification and found that it's pretty much a crap shoot whether or not the beads inhibit a given polymerase, and eluting from the beads is practically quantitative as long as you don't over dry them.
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