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

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

• 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