User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/06/06: Difference between revisions
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''' | |||
# | <!-- Precede each finished task with a checkmark ✓ --> | ||
* ✓ Transfections: H3me reporters into cell lines KAH126-1, 130-2, 132-8 | |||
* ✓ Polycomb-ATF negative control lines: colony plates | |||
---- | |||
'''Transfections'''<br> | |||
> U2OS Flp-in T-REx lines (puro/zeo/blast) + Polycomb-ATF plasmids (hygro)<br> | |||
--> Note: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in<br> | |||
> Use Lipofectamine, 6-well format<br> | |||
> Plates: | |||
# KAH126-1 Flp-in T-REx | |||
# KAH130-2 Flp-in T-REx | |||
# KAH132-8 Flp-in T-REx | |||
{| class="wikitable" border="0" cellspacing="5" <!-- Transfection table --> | |||
|- | |||
| <u>Plate-Well</u> || <u>Plasmid</u> || <u>DNA</u> || <u>Volume</u> || <u>Lipo</u> || <u>Opti-MEM (total)</u> | |||
|- | |||
| 1-1 || KAH96/V0201 + FlpE || 1.5 + 0.5 μg || 2.4 + 1.4 μL || 4 μL || 500 μL | |||
|- | |||
| 1-2 || KAH142/V0201 + FlpE || " || 2.4 + 1.4 μL || " || " | |||
|- | |||
| 1-3 || KAH146/V0201 + FlpE || " || 6.4 + 1.4 μL || " || " | |||
|- | |||
| 1-4 || KAH147/V0201 + FlpE || " || 9.6 + 1.4 μL || " || " | |||
|- | |||
| --- | |||
|- | |||
| 2-1 || KAH96/V0201 + FlpE || 1.5 + 0.5 μg || 2.4 + 1.4 μL || 4 μL || 500 μL | |||
|- | |||
| 2-2 || KAH142/V0201 + FlpE || " || 2.4 + 1.4 μL || " || " | |||
|- | |||
| 2-3 || KAH146/V0201 + FlpE || " || 6.4 + 1.4 μL || " || " | |||
|- | |||
| 2-4 || KAH147/V0201 + FlpE || " || 9.6 + 1.4 μL || " || " | |||
|- | |||
| --- | |||
|- | |||
| 3-1 || KAH96/V0201 + FlpE || 1.5 + 0.5 μg || 2.4 + 1.4 μL || 4 μL || 500 μL | |||
|- | |||
| 3-2 || KAH142/V0201 + FlpE || " || 2.4 + 1.4 μL || " || " | |||
|- | |||
| 3-3 || KAH146/V0201 + FlpE || " || 6.4 + 1.4 μL || " || " | |||
|- | |||
| 3-4 || KAH147/V0201 + FlpE || " || 9.6 + 1.4 μL || " || " | |||
|} | |||
> Add 16 μL (4x) Lipo to 1000 μL (4x) Opti-MEM --> R.T/ 5 min. <br> | |||
> Add DNA to 250 μL Opti-MEM <br> | |||
> Add 250 Lipo mix into each 250 DNA mix --> R.T./ 20 min. <br> | |||
> Add 500 μL complexes to each 3.5 mL well (4 ml med. total each); Grow cells at 37°C<br> | |||
> Refresh medium after 5 hours (ab-free)<br> | |||
> Grow for one/two days, then plate dilution cultures in selection medium (+hygro) | |||
---- | |||
'''Polycomb-ATF negative control lines: colony plates'''<br> | |||
> For each line make 2 plates: ~1x10<sup>5</sup> and 5x10<sup>4</sup> cells<br> | |||
> 25 ml 1 μg/mL puro, 15 μg/mL blast, 200 μg/mL zeo<br> | |||
> Also make 6-well back-up plate for all transfections (~3x10<sup>5</sup> cells in selection medium) | |||
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|} | |||
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Revision as of 09:47, 6 June 2013
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06/06/2013
Transfections
> Add 16 μL (4x) Lipo to 1000 μL (4x) Opti-MEM --> R.T/ 5 min.
Polycomb-ATF negative control lines: colony plates
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