Haynes Lab:Notebook/Jan/2015/12/04: Difference between revisions

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| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Vol
| bgcolor=#cfcfcf | Vol
| bgcolor=#cfcfcf | Mix* (x7)
| bgcolor=#cfcfcf | Mix* (x #)
| rowspan=7 | Expected:<br>1. CMV = 588
| rowspan=7 | Expected:<br>1. CMV = 588
| rowspan=7 | [[Image:somegel.jpg|150px|Hover name]]<br>5 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
| rowspan=7 | [[Image:somegel.jpg|150px|Hover name]]<br>5 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
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Note: *Prep one Mix per genomic DNA sample
Note: *Prep one Mix per template
* For each Aliquot 18.0 master mix into 6 tubes
* For each template, aliquot 18.0 master mix into 6 tubes


Thermal cycler: Labnet - GOTAQ
Thermal cycler: Labnet - GOTAQ

Revision as of 16:11, 3 December 2015

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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
  • Chromosensor 1
    • Validation Region 1
      • (1) chrsn1 f1/ chrsn1 r1
      • (2) chrsn1 f2/ chrsn1 r2
    • Validation Region 2
      • (3) chrsn1 f3/ chrsn1 r3
      • (4) chrsn1 f4/ chrsn1 r4
    • Validation Region 3
      • (5) chrsn1 f5/ chrsn1 r5
      • (6) chrsn1 f6/ chrsn1 r6
  • Chromosensor 2
    • Validation Region 1
      • (1) chrsn1 f1/ chrsn1 r1
      • (2) chrsn1 f2/ chrsn1 r2
    • Validation Region 2
      • (3) chrsn1 f3/ chrsn1 r3
      • (4) chrsn1 f4/ chrsn1 r4
    • Validation Region 3
      • (5) chrsn1 f5/ chrsn1 r5
      • (6) chrsn1 f6/ chrsn1 r6


  • 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
  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



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