[tatisaccon]

How important is to heat 5' Adapter in Step D of NEXTFLEX Small RNA-Seq Kit?

Hello!

How important is to heat 5' Adapter in Step D of NEXTFLEX® Small RNA-Seq Kit for Illumina® Platforms? In step D of the library preparation kit protocol I forgot to heat the 5 'adapter at 70C for 2 minutes and went straight to the incubation of 1h at 20 degrees C. The adapter 5 ' it's there, but it did not undergo this heating for 2 minutes. 12 samples in total. I wanted to know how much this can affect my final sample in sequencing, cause I do not have kit enough to do the 12 samples again.
If someone could share the experience with me would be great.

Thank you!!

[nucacidhunter]

Heating denatures the oligo self-dimers and possible secondary structure. Skipping heating will reduce the available adapter for ligation. Its impact will depend on ratio of adapter to template RNA. If you have done recommended number of PCR cycles and obtained expected yield then it has not affected the results.

[kayhand]

Hi,

Thanks for the info