User:Alexander S Mikheyev/Notebook/Midge genome/2010/04/16

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Contents

Trying Epicentre Illumina kit with midge DNA

Tagmentation

14 ulwater
1 ul1/3 dilution of 162ng/ul DNA extract
4 μl 5X Nextera LMW Reaction Buffer
1 μl Nextera Enzyme Mix (Illumina-compatible)
  • mix on ice
  • incubate 5 min @55C
  • purify with minelute kit from quiagen and elute into 10 ul buffer

PCR

17 μl Nuclease-Free water
5 μl Recovered DNA Fragment Library (from Step A3)
25 μl 2X Nextera PCR Buffer
1 μl 50X Nextera Primer Cocktail
1 μl 50X Nextera Adaptor 2*
1 μlNextera PCR Enzyme (2.5 U/μ
  • mix on ice
  • 72C 3 min; 95C 3min (95C 5s, 62C 30s, 72C 1min)x12, 72C 5 min
  • purified using quiagen pcr kit, using 2 washes
  • eluted in 50 ul, 6.5ng/ul

Gel

  • 3ul of non-purified PCR

Image:Asm100415a.png Image:Asm100415b.png

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