User:Alexander S Mikheyev/Notebook/P. rapida partial genome/2009/11/16

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Contents

precipitation

  • 0.5 volume (125 ul) of 7.5M NH4OAc, mix,
  • then add 2.5 volumes (950 ul) of room temperature 95% ethanol
  • 1 hour @ -20C
  • spin max speed 15 min
  • wash with 200 ul cold 80% ethanol
  • spin 5 min max
  • dry, re-suspend in 15 ul water
    • there is about 15 ul of gel left
    • looks like crap on the nanodrop (huge peak at 230)

PCR of ligated product

DNA (2 μl)
Phusion DNA polymerase (Finnzymes Oy) (25 μl)
PCR primer 1.1 (1 μl)
PCR primer 2.1 (1 μl)
Water (22 μl)
30s 98C (10s 98C; 30s 65C; 30s 72C)x10 5m 72C
2 ul of the product on a gel after cleaning
  • 6 ng/ul (61 fmol/ul) ==> 180 ng total yield
  • frozen at -20C
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