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

Alien genes

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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. DNA 10 4 fmol/ul linkers 2 1000 fmol/ul buffer 4 5x T4 ligase (Invitrogen) 1 1 U/il water 3 TOTAL 20 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