User:Karmella Haynes/Notebook/PcTF Genomics/2015/01/28: Difference between revisions

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(Autocreate 2015/01/28 Entry for User:Karmella_Haynes/Notebook/PcTF_Genomics)
 
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==mm/dd/yy==
==01/28/15==
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* Line item 1
* Cytology of KAH126


----
----
'''Line item 1'''<br>
'''Cytology of KAH126'''<br>
> Samples
* Last attempt at looking for Pc speckles in KAH-126 was at Harvard: https://www.openwetware.org/wiki/User:Karmella_Haynes/Notebook/Polycomb_project/2010/04/04
* Try again at different concentrations of dox induction (maybe over-expression gives high background?)


{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
'''Cell plating (1/23/15)'''
|-valign="top"
* Poly lysine coating of cells (done last week) - follow protocol
| <u>Reagent</u> || <u>Volume</u>
* Seed cells at 100k per well (aliquot in 1mL volumes into 24-well plate with poly-lysine coated cover slips on bottom)
|-
* Same day - Induce cells with 1.0, 0.1, and 0.01 μg/mL dox; final volume = 2 mL
| reagent 1 || # μL
** Tube 1: Added 8 μL 1 mg/mL dox to 4 mL growth medium (2x target concentration)
|-
** Tube 2: 0.5 mL from tube 1 into 4.5 mL medium (")
| reagent 2 || #
** Tube 3: 0.5 mL from tube 2 into 4.5 mL medium (")
|-
** Added 1 mL tube 1 into 1 mL cells in wells 1 & 2 (dilution by 1/2 --> reached target concentration of dox)
| reagent 3 || #
** Added 1 mL tube 2 into 1 mL cells in wells 3 & 4 (")
|-
** Added 1 mL tube 3 into 1 mL cells in wells 5 & 6 (")
| reagent 4 || #
** Wells 7 & 8 got 1 mL medium without dox
|-
| dH<sub>2</sub>O || #
|-
| &nbsp; || # μL
|}


--> Reaction conditions


'''Expression - next day (1/24/15)'''
* No signs of PC speckles
'''Expression - 5 days post induction (1/28/15)'''
* Neglected the cells, checked today out of curiosity; growth medium still healthy color
* A few cells show what look like Pc speckles in the nucleus; most others have diffuse RFP
* Fix and permeabilize these for IF staining - follow protocol




Revision as of 16:34, 28 January 2015

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01/28/15

  • Cytology of KAH126

Cytology of KAH126

Cell plating (1/23/15)

  • Poly lysine coating of cells (done last week) - follow protocol
  • Seed cells at 100k per well (aliquot in 1mL volumes into 24-well plate with poly-lysine coated cover slips on bottom)
  • Same day - Induce cells with 1.0, 0.1, and 0.01 μg/mL dox; final volume = 2 mL
    • Tube 1: Added 8 μL 1 mg/mL dox to 4 mL growth medium (2x target concentration)
    • Tube 2: 0.5 mL from tube 1 into 4.5 mL medium (")
    • Tube 3: 0.5 mL from tube 2 into 4.5 mL medium (")
    • Added 1 mL tube 1 into 1 mL cells in wells 1 & 2 (dilution by 1/2 --> reached target concentration of dox)
    • Added 1 mL tube 2 into 1 mL cells in wells 3 & 4 (")
    • Added 1 mL tube 3 into 1 mL cells in wells 5 & 6 (")
    • Wells 7 & 8 got 1 mL medium without dox


Expression - next day (1/24/15)

  • No signs of PC speckles


Expression - 5 days post induction (1/28/15)

  • Neglected the cells, checked today out of curiosity; growth medium still healthy color
  • A few cells show what look like Pc speckles in the nucleus; most others have diffuse RFP
  • Fix and permeabilize these for IF staining - follow protocol



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