User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/04/03

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Single rotifer extraction for population genetics

  • Trying to single rotifer-extractions using fusion primers designed for population genetics

PCR conditions

Reaction mixture conc. 1x 76
water 5.75 437
TopTaq buffer 10x 1 76
dNTP mix 10 mM each 0.2 15.2
Primer mix 5 uM each 1
TopTaq (QIAGEN) 5U/ul 0.05 3.8
template instagene 2
7
  • 3 min @95C, (5s 95C, 1 min 60C)x35 on Takara machine
  • using MIDs 1 through 12
  • rotifers from Gushikawa 01
    • each placed with 5-10 ul water into a PCR tube. Then 30ul instagene were added and incubated 30min at 56, 30min at 95C, vortexing occasionally

Results

  • Pretty much blank gel. There was some weak amplification in the last sample, but it looks like the extractions have failed.
  • Need to optimize single-rotifer extraction