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

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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

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



 * Single band at 1.2kb. Just the juno2 transpsoson made from the small quantity of DNA degraded during ligation?