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

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{| {{table}} cellspacing="7"  width=700px  
{| {{table}} cellspacing="7"  width=700px  
|-
|-
-
| <u>Plate-Well</u> || <u>Plasmid</u> || <u>DNA</u> || <u>Volume</u>|| dH2O || <u>Lipo</u>  || <u>Opti-MEM (total)</u>
+
| Plate-Well || Plasmid || DNA || Volume|| dH2O|| Lipo || PLUS reagent || <u>Opti-MEM (total)</u>
|-
|-
-
| 1-1            || BD002 (3:1) + FlpE  || 1.5 + 0.5 μg || 4.42 + 2.60 μL || 12.98μL|| 4 μL      || 500 μL
+
| 1-1            || BD002 (3:1) + FlpE  || 1.5 + 0.5 μg || 4.42 + 2.60 μL || 12.98μL|| 2.5μL|| 7.5 μL      || 570 μL
|-
|-
-
| 1-2            || BD002 (3:1) + FlpE || "          || 4.42 + 2.60 μL  || 12.98μL|| "             || "
+
| 1-2            || BD002 (3:1) + FlpE || "          || 4.42 + 2.60 μL  || 12.98μL|| " ||    "      || "
|-
|-
-
| 1-3            || BD002 (4:1) + FlpE || "          || 4.73 + 2.60 μL  || 12.67μL || "             || "
+
| 1-3            || BD002 (4:1) + FlpE || "          || 4.73 + 2.60 μL  || 12.67μL || "     ||  "    || "
|-
|-
-
| 1-4            ||  BD002 (4:1)+ FlpE || "          || 4.73 + 2.60 μL  || 12.67μL|| "             || "
+
| 1-4            ||  BD002 (4:1)+ FlpE || "          || 4.73 + 2.60 μL  || 12.67μL|| "     ||    "    || "
|-
|-
-
|1-5            || BD002 (3:1) + ---  ||           ||  4.42 + ---  || 15.58μL||       ||
+
|1-5            || BD002 (3:1) + ---  ||   "        ||  4.42 + ---  || 15.58μL||     "  || "||"
|-
|-
-
| 1-6            || BD002 (4:1) + ---  ||             ||  4.73 + ---  || 15.58μL ||     ||
+
| 1-6            || BD002 (4:1) + ---  ||   "          ||  4.73 + ---  || 15.58μL ||   "  ||  "||"
|}
|}

Revision as of 15:06, 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 dH2O Lipo PLUS reagent Opti-MEM (total)
1-1 BD002 (3:1) + FlpE 1.5 + 0.5 μg 4.42 + 2.60 μL 12.98μL 2.5μL 7.5 μL 570 μL
1-2 BD002 (3:1) + FlpE " 4.42 + 2.60 μL 12.98μL " " "
1-3 BD002 (4:1) + FlpE " 4.73 + 2.60 μL 12.67μL " " "
1-4 BD002 (4:1)+ FlpE " 4.73 + 2.60 μL 12.67μL " " "
1-5 BD002 (3:1) + --- " 4.42 + --- 15.58μL " ""
1-6 BD002 (4:1) + --- " 4.73 + --- 15.58μL " ""


> 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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