I am planning to sequence 3 mtDNA amplicons per sample using the Nextera XT kit. First I want to see if it is OK for the Nextera indexing be done once after the 3 amplicons are pooled in equal molar ratios?
Second many of my custom template primers have Tms below the 60-65C range outlined by illumnia. Just wondered if anyone has tried adding a CG tail on the 5' end of the primer (positioned between the primer and the adapter) to increase the Tm of the Custom template primer? My custom primers are AT rich and placed in universal regions, so increasing the length by adding sequence would be difficult.
Any advice most welcome ...
Second many of my custom template primers have Tms below the 60-65C range outlined by illumnia. Just wondered if anyone has tried adding a CG tail on the 5' end of the primer (positioned between the primer and the adapter) to increase the Tm of the Custom template primer? My custom primers are AT rich and placed in universal regions, so increasing the length by adding sequence would be difficult.
Any advice most welcome ...
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