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Old 06-18-2015, 11:11 AM   #1
Ingeneious
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Location: Los Angeles, CA

Join Date: Dec 2014
Posts: 30
Default End Repair + A-Tailing - how do they do it?

Some of the popular library prep kits out there (i.e. NEBNext Ultra and KAPA Hyper) are able to combine end repair and A-tailing into one step. I would like to do the same with minimal cost, but am not sure how they are doing it.

Both NEB and KAPA have the same thermocycler protocol:
  • 20C for 30 min (Activation?)
  • 65C for 30 min (Denaturation?)

End-repair can be done with some or a combination of the following enzymes:
  • DNA Polymerase I, Large (Klenow) Fragment (Active at 25C for 15 min)
  • T4 DNA Polymerase (Active at 15C for 12 min)
  • T4 Polynucleotide Kinase (Active at 37C for 30 min)

A-Tailing can be done with some or a combination of the following enzymes:
  • Taq Polymerase (Active at 72C for 20 min)
  • Klenow Fragment (3'→5' exo-) (Active at 37C for 30 min)

Anyone know how they do it?
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