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

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Alien genes
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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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To do

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

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

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