Smartseq Exonuclease
Hi all,
We have a few questions about using exonuclease before the preamp step of the Smartseq protocol.
1. Is this step intended for removing primer dimers, not excess single-stranded primers? Is that why lambda exonuclease (which works on dsDNA) is chosen instead of ExoI?
2. Wouldn't lambda exonuclease digest away the double-stranded cDNA template? If the ends got digested, the priming sites may be lost.
3. We have tried replacing lambda exonuclease with ExoI for a 30 min incubation, then an 85C, 15 min inactivation before adding primers for preamp step. However, yield is very low. Can somebody explain why?
4. Does anyone have the reference for the first suggestion of using lambda exonuclease?
Many thanks!
We have a few questions about using exonuclease before the preamp step of the Smartseq protocol.
1. Is this step intended for removing primer dimers, not excess single-stranded primers? Is that why lambda exonuclease (which works on dsDNA) is chosen instead of ExoI?
2. Wouldn't lambda exonuclease digest away the double-stranded cDNA template? If the ends got digested, the priming sites may be lost.
3. We have tried replacing lambda exonuclease with ExoI for a 30 min incubation, then an 85C, 15 min inactivation before adding primers for preamp step. However, yield is very low. Can somebody explain why?
4. Does anyone have the reference for the first suggestion of using lambda exonuclease?
Many thanks!
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