User:Alexander S Mikheyev/Notebook/rotifer alien genes/2009/11/15

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PCR of ligated product

Reagents 1x conc.
h2o 30 -
10x 5 10x
dNTP 2.5 10 mM
Linker_F 4 10 uM
Taq 0.4 5 U/um
DNA 8 4.4ng (~.45 fmol)/ul
TOTAL 50 Cocktail per vial
3m 93C (15s 93C; 15m 65C)x15
2 ul PCR product left lane
Gid 2009-11-16 13hr 22min.jpg

Rotifer DNA extraction

  • add 1ml of 1M NaCl to a 6 well dish filled with rotifers, they curl up
    • each well is about 10ml, resulting in a 100mM NaCl solution
  • spin at 1000g for 5 minutes
  • put into 1.5 ml tube, spin again
  • wash with small amount of water and go to extraction kit (quiagen micro)
  • yield 7ug for vaga and 5ug for ricciae
60 ng A. ricciae DNA, far right lane
Gid 2009-11-16 13hr 22min.jpg