User:Karmella Haynes/Notebook/Polycomb project/2013/01/09

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(Autocreate 2013/01/09 Entry for User:Karmella_Haynes/Notebook/Polycomb_project)
Current revision (21:29, 9 January 2013) (view source)
(01/09/13)
 
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==mm/dd/yy==
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==01/09/13==
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* Outline of experiments/ tasks
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* Thaw new cells: U2OS Flp-in TRex, HEK293 Flp-in TRex
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'''Task 1'''<br>
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'''Thaw cells'''<br>
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> Line item 1<br>
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* Quick thaw. Add to 7 mL FBS-tet-free
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* Spin for 3 min., 1000 rpm, r.t.
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{| class="wikitable" border="0" cellspacing="5"
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** U2OS Flp-in TRex - reuspend in McCoy's 5A/ 10% FBS/ 1% p/s
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** HEK293 Flp-in TRex - reuspend in DMEM / 10% FBS/ 1% p/s
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| <u>Reagent</u>  || <u>Volume</u> || <u>Conc.</u>
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* Grow in small flat bottom flasks
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| reagent 1      || vol 1 || conc 1
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| reagent 2      || vol 2 || conc 2
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| dH<sub>2</sub>O || vol 3 || &nbsp;
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| &nbsp;          || total vol || &nbsp; --> Reaction conditions
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|}
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----
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'''Lipo transfection'''<br>
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> Details
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{| class="wikitable" border="0" cellspacing="5" <!-- Lipofectamine transfection table -->
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|-
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| <u>Wells</u> || <u>Plasmid</u> || <u>DNA</u> || <u>Volume</u> || <u>Lipo</u>  || <u>Opti-MEM (total)</u>
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| 1            || name          || 2 μg      || ### μL        || 4 μL          || 500 μL
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| 2            || name          || "          || ### μL        || "            || "
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| 3            || name          || "          || ### μL        || "            || "
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| 4            || name          || "          || ### μL        || "            || "
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| 5            || name          || "          || ### μL        || "            || "
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| 6            || name          || "          || ### μL        || "            || "
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|}
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> Add Lipo to 250 μL Opti-MEM --> R.T/ 5 min. <br>
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> Add DNA to 250 μL Opti-MEM <br>
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> Add DNA mix to Lipo mix --> R.T./ 20 min. <br>
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> '''Remove 500 μL medium from each well'''; Add 500 μL complexes to each well (4 ml med. each); Grow cells at 37&deg;C<br>
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> Refresh medium after 5 hours (ab-free)<br>
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----
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'''Fugene transfection'''<br>
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> Details<br>
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{| class="wikitable" border="0" cellspacing="5" <!-- Fugene transfection table -->
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|-
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| <u>Wells</u> || <u>Plasmid</u> || <u>DNA</u> || <u>Volume</u> || <u>Fugene</u> || <u>Opti-MEM</u>
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|-
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| 1-6          || name          || 200 ng    || ### μL        || 1.8 μL        || 18.2 μL
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| 7-12        || name          || "          || ### μL        || "            || "
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| 13-18        || name          || "          || ### μL        || "            || "
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| 19-24        || name          || "          || ### μL        || "            || "
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|}
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> Master mixes (4, x6): 10.8 μL Fugene + 109.2 μL Opti-MEM --> R.T/ 5 min. <br>
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> Add 6x vol DNA --> R.T./ 20 min. <br>
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> Add 20 μL complexes to each well (1 ml med. each); Grow cells at 37&deg;C overnight<br>
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01/09/13

  • Thaw new cells: U2OS Flp-in TRex, HEK293 Flp-in TRex

Thaw cells

  • Quick thaw. Add to 7 mL FBS-tet-free
  • Spin for 3 min., 1000 rpm, r.t.
    • U2OS Flp-in TRex - reuspend in McCoy's 5A/ 10% FBS/ 1% p/s
    • HEK293 Flp-in TRex - reuspend in DMEM / 10% FBS/ 1% p/s
  • Grow in small flat bottom flasks


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