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

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
091107nano.JPG

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

Gid 2009-11-08 12hr 19min.jpg

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