User:Alexander S Mikheyev/Notebook/rotifer alien genes/2009/11/05

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Contents

To do

  • precipitate probe and end repaired reaction
  • ligate adapters to p. rapida DNA
  • order transposase enzymes

Juno2 probe preparation

make 7.5M NH4OAC

  • 867 milligrams NH4OAC in 1.5 ml water for 7.5M solution

precipitation

  • 0.5 volume (25 ul) of 7.5M NH4OAc, mix,
  • then add 2.5 volumes (188 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 30 ul water
    • 297ng/ul ssDNA
    • 0.46 pmol/ul

P. rapida adapter ligation

precipitate end-repaired reaction

  • 0.5 volume (35 ul) of 7.5M NH4OAc, mix,
  • then add 2.5 volumes (263 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 30 ul water
    • 36 ng/ul DNA
    • ~4 fmol/ul

ligation

Reagents 1x conc.
DNA104 fmol/ul
linkers21000 fmol/ul
buffer45x
T4 ligase (Invitrogen)11 U/il
water3
TOTAL20
  • overnight @14C, in at 13:40

Making PEG for size selective DNA precipitation

  • 150 mg PEG 8000
  • 350 ul 25mM MgCl2 (Invitrogen stock for PCR)
  • store @4C
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