User:Alexander S Mikheyev/Notebook/rotifer alien genes/2009/11/06

Alien genes

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To do PCR of ligated samples Hybridization? Feed rotifer cultures Isolating P. roseola Juno2 Getting rid of unligated adapters by PEG precipitation add 60 ul TE and 40 ul PEG to 20 ul ligation reaction, mix centrifuge at max for 15 min @ rt remove supernatant, wash with 30 ul 80% ethanol, dry re-dissolve in 30 ul water 4.4 ng/ul (36% yield) 0.45 fmol/ul PCR of ligated product Reagents 1x conc. h2o 15.05 - 10x 2.5 10x dNTP 1.25 10 mM Linker_F 2 10 uM Taq 0.2 5 U/um DNA 4 4.4ng (~.45 fmol)/ul TOTAL 25 Cocktail per vial 3m 93C (15s 93C; 15m 65C)x10 Precipitation 1 volume (28 ul) PEG 15 min at 0C spin 15 min max wash with 80% ethanol dissolve in 20 ul water 9 ng/ul Hybridization the probe has a Tm of 98C at 1M NaCl, I want to hybridize things that may mismatch by as much as 30%, hence the low hybridization temperature Reagents 1x conc. total h2o 29.5 - DNA 20 .9 fmol/ul 18 fmol probe 0.5 0.46 pmol/ul 250 fmol 12xSSC, .1%SDS 50 TOTAL 100 Program: 10’ at 95C. 85oC for 1’ 85oC and step down 0.2C every 10 seconds (5 step downs per degree x 20 degrees = 99 cycles) stay at 65C for 16 hours ramp down to 4C for infinity