User:Alexander S Mikheyev/Notebook/P. rapida partial genome/2009/11/13

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Contents

To Do

  • add +A onto end-repaired sample
  • set up overnight ligation

+A addition

  • end-repaired sample with Invitrogen PureLink PCR Purification Kit, eluting in 35 μl of buffer
  • 17ng/ul, or about 4 pmol total DNA @ 200bp
DNA sample (32 ul)
1 mM dATP (10 ul)
10x Klenow buffer (5 ul)
Klenow exo (3’ to 5’ exo minus) (3 ul)
  • Incubate for 30 minutes at 37°C.
  • MinElute PCR Purification Kit to purify on one QIAquick MinElute column, eluting in 10 μl of EB.

Adapter ligation

DNA sample (10 ul)
water (5 ul)
2x ligation buffer (25 ul)
adapters @ 15uM (5 ul)
ligase (5 ul)
  • 16C for overnight
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