User:Alexander S Mikheyev/Notebook/rotifer alien genes/2009/11/19

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To do

  • Oil flies
  • prepare for sequencing
  • Order:
    • GITC
  • Submit AmNat paper

PEG precipitation

  • recipe:
    • 3g PEG 8000
    • 2.19g NaCl
    • Bring up to 15ml with good water.
  • added 8ul PEG to PCR
  • spun at max speed for 15 min
  • removed PEG, added 150ul cold 80% ethanol
  • spin 7 min
  • remove ethanol, dry
  • re-suspend in 10 ul water
  • nanodrop
Sample Nucleic Acid Conc. Unit 260/280 260/230
intergenic 12 ng/µl 1.95 0.62
AV10007 11.2 ng/µl 1.85 0.67
AV10016 13.6 ng/µl 1.81 0.67
AV10027 10.2 ng/µl 1.95 0.88
AV10153 8 ng/µl 2.34 1.53
PR10008 13.8 ng/µl 1.69 2.18
  • somehow I don't feel completely comfortable with potential effects of CoraLoad dye on precipitation, so I'll run a gel:
Gid 2009-11-19 13hr 58min.jpg

InstaGene matrix for extraction of small numbers of rotifers

  • placed 1, 2, 4 and 8 A. ricciae into 30 ul instagene buffer
  • 30 min@ 56, with one vortex, 8 min at 99C
Reagents 1x conc. 9
h2o 4.75 - 42.75
10x 1 10x 9
dye 1 10x 9
dNTP 0.2 10 mM 1.8
Intergenic F 1 10 uM ea 9
Taq 0.05 5 U/um 0.45
DNA 2 20 ng/ul
TOTAL 10 Cocktail per vial 8
  • 1ul and 2 ul extraction
1 rotifer 2 rotifers 4 rotifers 8 rotifers
  • 3m 94C (10s 94C; 3m 60C)x35
  • try one set with 1 ul DNA, and another set with 2 ul DNA
  • 3ul PCR product 2ul ladder
  • 1ul 4 rotifer lane -- no sample loaded into PCR
Gid 2009-11-19 18hr 17min.jpg