Haynes Lab:Notebook/Jan/2015/12/04

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Entry title

  • PCR - validation of Chromatin sensor insert



PCR - validation of Chromatin sensor insert

  • Designed primers to amplify three overlapping regions across the transgenes: Chromosensor1, Chromosensor 2
  1. chrsn1 f1/ chrsn1 r1; Validation region 1; sensor 1 (and 2)
  2. chrsn1 f2/ chrsn1 r2; Validation region 1; sensor 1 (and 2)


  • Round 1: Control templates to test primer function
  1. U-2 OS genomic DNA (neg)
  2. Chromosensor 1 plasmid DNA
  3. Chromosensor 2 plasmid DNA


  • Round 2: Genomic DNA prepped from candidate clonal lines
    • To be done after chosing a single primer pair per validation region
  1. BD###, Chromosensor 1
  2. BD###, Chromosensor 1
  3. BD###, Chromosensor 2
  4. BD###, Chromosensor 2


ROUND 1 PCR: Neg, pos controls

Reagent Vol Mix* (x #) Expected:
1. CMV = 588
Hover name
5 μL/lane; 1% agarose; Ladder
genomic OR plasmid DNA 1.0 / 0.5 7.0 / 4.5
10 μM forward primer 1.0 ---
10 μM reverse primer 1.0 ---
2x GoTaq green 10.0 70.0
dH2O 7.0 / 7.5 49.0 / 52.5
  20.0 μL ---

Note: *Prep one Mix per template

  • For each template, aliquot 18.0 master mix into 6 tubes

Thermal cycler: Labnet - GOTAQ

  • 95°C 3 min.
  • 30x[95°C, 30 sec; 57°C 30 sec; 72°C 30 sec]
  • 72°C 3 min.
  • 4°C, ∞


  • Conclusions