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

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



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


 * 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