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

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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
091106nanopr.JPG

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

Gid 2009-11-06 14hr 26min.jpg

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
091106nanopr2.JPG

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