User:Alexander S Mikheyev/Notebook/rotifer alien genes/2009/11/04

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Juno2 isolation

Gel extraction of P. rapida DNA, continued from 03 Nov

  • make 10 ml 7.5M NH4OAc
  • 0.5 volume (8.5 ml) of 7.5M NH4OAc, mix,
  • then add 2.5 volumes (64 ml) of room temperature 95% ethanol
  • let stand 2 hours 11am-
  • split into two tubes (34 ml each) andspin 15 min at 15000 rcs
  • ultimately, dissolved in ~177 ul of water @ ~31 ng/ul, with some agarose remaining. About 5μg total yield
Nanodrop reading 3μl log2 ladder, 3μl sample
091104nano.GIF
Gid 2009-11-04 16hr 53min.jpg
  • concentrated using a microcon kit
    • 19 min @ 0.5k g, checking every 3 min
    • invert tube, 3 min @ 1k g
    • 47 μl final volume

Blunt end repair of P.rapida DNA

Reagents 1x conc.
DNA 47.5 133ng/ul
10x 7 10x
ATP 7 10 mM
[|NEB blunting enzyme] 1.5
dNTPs 7 10 mM
TOTAL 70
Hold 16 hours at 16C deactivate 10 min @65C program exp003 on PCR
  • however, skipped the denaturing step and continued to precipitation