This is a quick question with hopefully a quick answer
Illumina published a recommended protocol for 16S Metagenomic Sequencing Library Preparation. In the protocol they suggest that you can swap in your own primers, just add on the appropriate overhang sequences.
Their bulletin on this protocol says..."Locus-specific portion of the forward and reverse primers (NOT including the overhang sequence) must have a melting temperature (Tm) of 60°C to 65°C. If melting temperature is lower than 60°C, Tm can be increased by adding several “padding” nucleotides."
Why would this be? Does this mean that the overhang adapters interfere with primers that amplify at conditions other than what is recommended?
I'm interested in using this protocol with the primers used by Mahe et al 2015, Comparing High‐throughput Platforms for Sequencing the V4 Region of SSU‐rDNA in Environmental Microbial Eukaryotic Diversity Surveys
Illumina published a recommended protocol for 16S Metagenomic Sequencing Library Preparation. In the protocol they suggest that you can swap in your own primers, just add on the appropriate overhang sequences.
Their bulletin on this protocol says..."Locus-specific portion of the forward and reverse primers (NOT including the overhang sequence) must have a melting temperature (Tm) of 60°C to 65°C. If melting temperature is lower than 60°C, Tm can be increased by adding several “padding” nucleotides."
Why would this be? Does this mean that the overhang adapters interfere with primers that amplify at conditions other than what is recommended?
I'm interested in using this protocol with the primers used by Mahe et al 2015, Comparing High‐throughput Platforms for Sequencing the V4 Region of SSU‐rDNA in Environmental Microbial Eukaryotic Diversity Surveys
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