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

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<!-- Precede each finished task with a checkmark &#x2713; -->
<!-- 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
+
* &#x2713; Transfections: H3K27me3 reporters into cell lines BD002 (KAH201+KAH182+V0200)
* &#x2713; Polycomb-ATF negative control lines: colony plates
* &#x2713; Polycomb-ATF negative control lines: colony plates
Line 17: Line 17:
----
----
'''Transfections'''<br>
'''Transfections'''<br>
-
> U2OS Flp-in T-REx lines (puro/zeo/blast) + Polycomb-ATF plasmids (hygro)<br>
+
> U2OS Flp-in T-REx lines (puro/zeo/blast) + FRT-marked mammalian transfection vector (hygro)<br>
--> Note: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in<br>
--> Note: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in<br>
> Use Lipofectamine, 6-well format<br>
> Use Lipofectamine, 6-well format<br>
> Plates:
> Plates:
-
# KAH126-1 Flp-in T-REx
+
# BD002 (3:1) Flp-in T-REx
-
# KAH130-2 Flp-in T-REx
+
#BD002 (4:1) Flp-in T-REx
-
# KAH132-8 Flp-in T-REx
+
 
 +
BD002 (3:1) = 339 ng/μl
 +
BD002 (4:1) = 317 ng/μl
 +
PlpE = 192 ng/μl
 +
 
Line 30: Line 34:
| <u>Plate-Well</u> || <u>Plasmid</u> || <u>DNA</u> || <u>Volume</u> || <u>Lipo</u>  || <u>Opti-MEM (total)</u>
| <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
+
| 1-1            || BD002 (3:1) + FlpE  || 1.5 + 0.5 μg || 4.42 + 2.60 μL || 4 μL      || 500 μL
|-
|-
-
| 1-2            || KAH142/V0201 + FlpE || "          || 2.4 + 1.4 μL  || "            || "
+
| 1-2            || BD002 (3:1) + FlpE || "          || 4.42 + 2.60 μL  || "            || "
|-
|-
-
| 1-3            || KAH146/V0201 + FlpE || "          || 6.4 + 1.4 μL   || "            || "
+
| 1-3            || BD002 (4:1) + FlpE || "          || 4.73 + 2.60 μL || "            || "
|-
|-
-
| 1-4            || KAH147/V0201 + FlpE || "          || 9.6 + 1.4 μL  || "            || "
+
| 1-4            || BD002 (4:1)+ FlpE || "          || 4.73 + 2.60 μL  || "            || "
|-
|-
-
| ---
+
|1-5            || BD002 (3:1) + ---              ||   4.42 + --- ||       ||
-
|-
+
-
| 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  || "            || "
+
|-
|-
 +
|1-6            || BD002 (4:1) + ---              ||  4.73 + ---    ||      ||
| ---
| ---
-
|-
+
 
-
| 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 16 μL (4x) Lipo to 1000 μL (4x) Opti-MEM --> R.T/ 5 min. <br>

Revision as of 14:43, 6 June 2013

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

  • ✓ Transfections: H3K27me3 reporters into cell lines BD002 (KAH201+KAH182+V0200)
  • ✓ Polycomb-ATF negative control lines: colony plates



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

  1. BD002 (3:1) Flp-in T-REx
  2. BD002 (4:1) Flp-in T-REx

BD002 (3:1) = 339 ng/μl BD002 (4:1) = 317 ng/μl PlpE = 192 ng/μl


Plate-Well Plasmid DNA Volume Lipo Opti-MEM (total)
1-1 BD002 (3:1) + FlpE 1.5 + 0.5 μg 4.42 + 2.60 μL 4 μL 500 μL
1-2 BD002 (3:1) + FlpE " 4.42 + 2.60 μL " "
1-3 BD002 (4:1) + FlpE " 4.73 + 2.60 μL " "
1-4 BD002 (4:1)+ FlpE " 4.73 + 2.60 μL " "
1-5 BD002 (3:1) + --- 4.42 + ---
1-6 BD002 (4:1) + --- 4.73 + --- ---


> 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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