User:Alexander S Mikheyev/Notebook/rotifer alien genes/2009/11/07

Alien genes

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B&W buffer 60 mg Tris base 10 mg EDTA 2.9g NaCl adjust pH to 7.5 Hybridization Dynabeads (10 mg/ml) can bind 200 pmol ssDNA per mg we have .25 dsDNA in the oligo, so only need 12.5 ul of the dynabeads. We will live a little ant take 100 ul wash 3x with 100 ul B&W buffer re-suspend in 100 ul B&W buffer add hybridization reaction from yesterday 2h at 33C to bind oligos to beads Percent identity for washes with different concentrations of SSC and temperatures assuming 1% mismatch lowers temperature by 1.4 C Tm probe 90 85 68 SSC 2x 1x .1x Temp (C) [Na+] M 0.33 0.165 0.0165 24 52.9 56.4 68.6 40 64.3 67.9 80.0 50 71.4 75.0 87.1 60 78.6 82.1 94.3 Washes (at room temperature) set heating block to 95C (for later) Twice for two minutes 100 ul 2x SSC 0.1% SDS (50% identity) ==> 100 ul 20x SSC, 100 ul 1% SDS, in 800 ul water Twice for two minutes 100 ul 0.1x SSC 0.1% SDS (70% identity) ==> 50 ul 2x SSC in 100 ul 1%SDS in 850 ul water DNA collection and concentration add 200 ul quiagen elution buffer incubate 5 min @ 95C, collect supernatant concentrate using microcon tubes into about 30 ul spectrum is not really informative PCR of enriched 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 unknown TOTAL 25 Cocktail per vial 3m 93C (15s 93C; 15m 65C)x35 Single band at 1.2kb. Just the juno2 transpsoson made from the small quantity of DNA degraded during ligation?