User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/06/06

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=Date=
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==06/06/2013==
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'''List title'''
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# List items
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<!-- Precede each finished task with a checkmark &#x2713; -->
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* &#x2713; Transfections: H3me reporters into cell lines KAH126-1, 130-2, 132-8
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* &#x2713; Polycomb-ATF negative control lines: colony plates
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----
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'''Transfections'''<br>
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> U2OS Flp-in T-REx lines (puro/zeo/blast) + Polycomb-ATF plasmids (hygro)<br>
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--> Note: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in<br>
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> Use Lipofectamine, 6-well format<br>
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> Plates:
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# KAH126-1 Flp-in T-REx
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# KAH130-2 Flp-in T-REx
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# KAH132-8 Flp-in T-REx
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{| class="wikitable" border="0" cellspacing="5" <!-- Transfection table -->
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|-
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| <u>Plate-Well</u> || <u>Plasmid</u> || <u>DNA</u> || <u>Volume</u> || <u>Lipo</u>  || <u>Opti-MEM (total)</u>
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|-
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| 1-1            || KAH96/V0201 + FlpE  || 1.5 + 0.5 μg || 2.4 + 1.4 μL || 4 μL      || 500 μL
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|-
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| 1-2            || KAH142/V0201 + FlpE || "          || 2.4 + 1.4 μL  || "            || "
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|-
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| 1-3            || KAH146/V0201 + FlpE || "          || 6.4 + 1.4 μL  || "            || "
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|-
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| 1-4            || KAH147/V0201 + FlpE || "          || 9.6 + 1.4 μL  || "            || "
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|-
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| ---
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|-
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| 2-1            || KAH96/V0201 + FlpE  || 1.5 + 0.5 μg || 2.4 + 1.4 μL || 4 μL      || 500 μL
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|-
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| 2-2            || KAH142/V0201 + FlpE || "          || 2.4 + 1.4 μL  || "            || "
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|-
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| 2-3            || KAH146/V0201 + FlpE || "          || 6.4 + 1.4 μL  || "            || "
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|-
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| 2-4            || KAH147/V0201 + FlpE || "          || 9.6 + 1.4 μL  || "            || "
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|-
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| ---
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|-
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| 3-1            || KAH96/V0201 + FlpE  || 1.5 + 0.5 μg || 2.4 + 1.4 μL || 4 μL      || 500 μL
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|-
 +
| 3-2            || KAH142/V0201 + FlpE || "          || 2.4 + 1.4 μL  || "            || "
 +
|-
 +
| 3-3            || KAH146/V0201 + FlpE || "          || 6.4 + 1.4 μL  || "            || "
 +
|-
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| 3-4            || KAH147/V0201 + FlpE || "          || 9.6 + 1.4 μL  || "            || "
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|}
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> Add 16 μL (4x) Lipo to 1000 μL (4x) Opti-MEM --> R.T/ 5 min. <br>
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> Add DNA to 250 μL Opti-MEM <br>
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> Add 250 Lipo mix into each 250 DNA mix --> R.T./ 20 min. <br>
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> Add 500 μL complexes to each 3.5 mL well (4 ml med. total each); Grow cells at 37&deg;C<br>
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> Refresh medium after 5 hours (ab-free)<br>
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> Grow for one/two days, then plate dilution cultures in selection medium (+hygro)
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 +
 
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----
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'''Polycomb-ATF negative control lines: colony plates'''<br>
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> For each line make 2 plates: ~1x10<sup>5</sup> and 5x10<sup>4</sup> cells<br>
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> 25 ml 1 μg/mL puro, 15 μg/mL blast, 200 μg/mL zeo<br>
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> 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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<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
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|}
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__NOTOC__

Revision as of 11:47, 6 June 2013

PcTF Subcloning in E. coli Main project page
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06/06/2013

  • ✓ Transfections: H3me reporters into cell lines KAH126-1, 130-2, 132-8
  • ✓ Polycomb-ATF negative control lines: colony plates



Transfections
> U2OS Flp-in T-REx lines (puro/zeo/blast) + Polycomb-ATF plasmids (hygro)
--> Note: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in
> Use Lipofectamine, 6-well format
> Plates:

  1. KAH126-1 Flp-in T-REx
  2. KAH130-2 Flp-in T-REx
  3. KAH132-8 Flp-in T-REx


Plate-Well Plasmid DNA Volume Lipo Opti-MEM (total)
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.
> Add DNA to 250 μL Opti-MEM
> Add 250 Lipo mix into each 250 DNA mix --> R.T./ 20 min.
> Add 500 μL complexes to each 3.5 mL well (4 ml med. total each); Grow cells at 37°C
> Refresh medium after 5 hours (ab-free)
> Grow for one/two days, then plate dilution cultures in selection medium (+hygro)



Polycomb-ATF negative control lines: colony plates
> For each line make 2 plates: ~1x105 and 5x104 cells
> 25 ml 1 μg/mL puro, 15 μg/mL blast, 200 μg/mL zeo
> Also make 6-well back-up plate for all transfections (~3x105 cells in selection medium)



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