Jessica Karen Wong/Notebook/2007-7-5

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To Do

  • PCR clean and then Digest T9002 with Nsi1
  • Run gel of I2056
  • Drop off sequencing


  • Heat Shocked Mfe1 digest
  • PCR cleaned, DNA concentration was only 19 ng/ul
  • Continued the sequential digest
    • Used all the Mfe1 digest product (30ul), 12.5 ul water, buffer 3
  • Also trying a double digest of Mfe1 and Nsi1 in buffer 2
    • 11ul scarred DNA, 31.5 ul water
  • PCR cleaned both
    • Sequential Digest DNA concentration 10.9 ng/ul
    • Double Digest DNA concentration 19 ng/ul


  • Ran gel of the overnight colony PCR
  • Have a faint band of the right size
  • Overnighted 2 cultures
    • One for glycerol and sequencing, one to miniprep for scarring
    • Used 5ul Amp and 10ul Tet per 5ml culture


Gel from 7/5/07
Gel from 7/5/07
  • Did a 100ul preparatory PCR at 53.5
  • Fwd primer BB_E0240_F was 38.2nmole added 956ul water
  • Rev primer BB_backbone was 38.09nmole added 952ul water
  • Ran a gel of the PCR, sample to the right of 2 log ladder
    • Faint band of correct size (3kb) but much brighter band that was too small (1kb)
  • Didn't use a long enough elongation time
  • Will retry with longer elongation time and both vent and phusion polymerases
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