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Hi all,
I’m trying to do cleanup reactions for my PCR’s with the protocol. When I go with a ratio of 2, the protocol works fine with any okayish yield of about 60-70% (10 uL PCR product, 10 uL Beads, 5 uL water, 50 uL of binding buffer).
However when I lower the ratio the yield directly drops to ~ 10%. I assume that it is due to the pH of the PCR buffer. I’m using the Qiagen Multiplex Plus kit which is buffered at pH 8.7. When adding the binding buffer the color does not stay yellow but changes to violet again, which let’s me assume that I don’t have to correct pH for binding the DNA. I tried giving the binding buffer a higher buffer capacity by raising the Bis-Tris to 20 mM but it does not help, it even seems to worsen the problem. Anyone else is having this struggles and maybe knows a solution to this? When testing different ratios with a ladder I had great results with yields up to 90%.
best Dominik
Ahoy,
Way, way late so I truly hope you have found a solution by now.
If not, how about you dilute your PCR product prior to adding the binding buffer?
This way the binding solution buffer capacity should overcome the PCR mix buffer capacity.e.g. rather than using 10ul PCR product and 15ul of Binding buffer (1.5 ratio)
you could use
10 ul PCR product + 20 ul H2O and then add 45ul of Binding Buffer (still 1.5 ratio).
And during the final elution you can reduce your volume to 15-20ul once more.
The technique relies on buffer concentration and not the Bead to DNA ratio so you would not even have to use more beads.
Cheers
Tim M
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