Which “High Fidelity PCR Master Mix” would you recommend using with the CleanTag Ligation Kit for Small RNA Library Prep? I noticed your protocol uses more than the volume that comes with the TruSeq Small RNA kit, so I was wondering which PCR MasterMix have you found most success with? -Steve

Posted on by TriLink BioTech

Hi Steve,

Thank you for your question. We recommend the Q5® High Fidelity 2x Master Mix (catalog # M0492S) from NEB for use with the CleanTag™ Ligation Kit for Small RNA Library Prep. You can view our full list of recommended reagents here.

Please let me know if you have any other questions.

Best regards,
Elizabeth

I would like to block ligation at the 3′ end of an oligo. The traditional way to block described in the literature is C3 spacer. I was wondering if I label the 3′ end of an oligo with the Cy5 dye, would the dye molecule effectively block ligation? -Chun-Nan

Posted on by TriLink BioTech

Dear Chun-Nan,

The blocking groups most often described in the literature are 3′ dideoxy-C, 3′ C3 Spacer (C3-OH) or 3′ Amino Linker (C6-NH2). All the mentioned modifications permanently block the 3′ hydroxyl group of the 3′ base. We can use the 3′ Amino Linker and label it with the Cyanine 5 dye which would continue to block the 3′ hydroxyl group.

Please contact us if you have any other questions.

Best regards,
Sabrina Shore, MS
Scientist, Research & Development