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

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==Entry title==
==Entry title==
* Insert content here...
* PCR - validation of Chromatin sensor insert
 
 
----
 
'''PCR - validation of Chromatin sensor insert'''
 
* Designed primers to amplify three overlapping regions across transgenes: Chromosensor1, Chromosensor 2
* Chromosensor 1
** Validation Region 1
*** chrsn1 f1/ chrsn1 r1
*** chrsn1 f2/ chrsn1 r2
** Validation Region 1
** Validation Region 1
 
** 2x 50 μL rxns
** Template = CMV (KAH
** f primer: XbaI-CMV f1 - 5'-CCTTTCTAGAGTTGACATTGATTATTGGCTAG
** r primer: SacII-NS-CMV r1 - 5'-CATTCCGCGGGCGGCCGCTACTAGTGAGCTCTGC
 
 
{| {{table}} cellspacing="3" <!-- PCR rxn table -->
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Vol
| bgcolor=#cfcfcf | Mix (x2)
| rowspan=7 | Expected:<br>1. CMV = 588
| rowspan=7 | [[Image:KAH051115_gel1.jpg|150px|Hover name]]<br>5 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
| plasmid DNA || 0.5  || 1.0
|-
| 10 μM XbaI-CMV f1    || 1.0  || 2.0 
|-
| 10 SacII-NS-CMV r1  || 1.0  || 2.0
|-
| 2x GoTaq green    || 25.0 || 50.0
|-
| dH<sub>2</sub>O    || 22.5  || 45.0
|-
| &nbsp;            || 50.0 μL ||  ---
|}
 
Thermal cycler: Labnet - GOTAQ
* 95°C 3 min.
* 30x[95°C, 30 sec; 55°C 30 sec; 72°C 30 sec]
* 72°C 3 min.
* 4°C, ∞
 
 
* Conclusions




Revision as of 15:41, 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 transgenes: Chromosensor1, Chromosensor 2
  • Chromosensor 1
    • Validation Region 1
      • chrsn1 f1/ chrsn1 r1
      • chrsn1 f2/ chrsn1 r2
    • Validation Region 1
    • Validation Region 1
    • 2x 50 μL rxns
    • Template = CMV (KAH
    • f primer: XbaI-CMV f1 - 5'-CCTTTCTAGAGTTGACATTGATTATTGGCTAG
    • r primer: SacII-NS-CMV r1 - 5'-CATTCCGCGGGCGGCCGCTACTAGTGAGCTCTGC


Reagent Vol Mix (x2) Expected:
1. CMV = 588 		Hover name
5 μL/lane; 1% agarose; Ladder
plasmid DNA 0.5 1.0
10 μM XbaI-CMV f1 1.0 2.0
10 SacII-NS-CMV r1 1.0 2.0
2x GoTaq green 25.0 50.0
dH2O 22.5 45.0
  50.0 μL ---

Thermal cycler: Labnet - GOTAQ

  • 95°C 3 min.
  • 30x[95°C, 30 sec; 55°C 30 sec; 72°C 30 sec]
  • 72°C 3 min.
  • 4°C, ∞


  • Conclusions



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