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

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(05/06/10)
Current revision (15:39, 19 June 2013) (view source)
(06/06/2013)
 
Line 63: Line 63:
BD002 (3:1) = 339 ng/μl
BD002 (3:1) = 339 ng/μl
BD002 (4:1) = 317 ng/μl
BD002 (4:1) = 317 ng/μl
-
PlpE = 192 ng/μl
+
KAH187 = 441.5 ng/μl
 +
FlpE = 192 ng/μl
{| {{table}} cellspacing="7"  width=700px  
{| {{table}} cellspacing="7"  width=700px  
Line 71: Line 72:
| 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
| 1-1            || 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 (4:1) + FlpE || "          || 4.73 + 2.60 μL  || 12.67μL|| "  ||    "      || "
|-
|-
-
| 1-3           || BD002 (4:1) + FlpE || "          || 4.73 + 2.60 μL  || 12.67μL || "    ||  "    || "
+
| 1-3 (-) Ctrl    || BD002 (3:1) + --- || 2 μg      || 4.42 + --- μL  || 15.58μL || "    ||  "    || "
|-
|-
-
| 1-4           ||  BD002 (4:1)+ FlpE || "          || 4.73 + 2.60 μL  || 12.67μL|| "    ||    "    || "
+
| 1-4 (-)Ctrl      ||  --- + --- || 0 μg        || --- + --- μL  || 20.0μL|| "    ||    "    || "
|-
|-
-
|1-5            || BD002 (3:1) + ---  ||  "        ||  4.42 + --- || 15.58μL||    "  || "||"
+
|1-5            || KAH187    ||  20 μg        ||  4.53 || 15.46μL||    "  || "||"
-
|-
+
-
| 1-6            || BD002 (4:1) + ---  ||  "          ||  4.73 + ---  || 15.58μL ||    "  ||  "||"
+
|}
|}
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__NOTOC__
__NOTOC__

Current revision

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

  • ✓ Minipreps: FlpE (4)

Minipreps
> Check with E, P, and E/P digests

Reagent 1,4,7,10 2,5,8,11 3,6,9,12 Expected:
1. FlpE, E = 7160, 546
2. FlpE, P = 7706
3.FlpE, E/P=5649, 1511, 546
E/P digests 05/06/10
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 2.0 2.0
10X buffer 1.5 1.5 1.5
EcoRI 1.0 --- 1.0
PstI --- 1.0 1.0
dH2O 10.5 10.5 9.5
 
 
 
 

15 μL --> 37°C/ ~15 min.


--> Conclusion: Success! Combine 4 preps (300μL total) for DNA clean-up (tomorrow)
--> Streak clone (saved toothpick) onto 100 μg/mL Amp plate.


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



1 day before transfection:

  1. Seed cultures in 6-well plates at ~2.5x105 so that cells will be ~5.0x105 at the time of transfection.

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 KAH187 = 441.5 ng/μl FlpE = 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 (4:1) + FlpE " 4.73 + 2.60 μL 12.67μL " " "
1-3 (-) Ctrl BD002 (3:1) + --- 2 μg 4.42 + --- μL 15.58μL " " "
1-4 (-)Ctrl --- + --- 0 μg --- + --- μL 20.0μL " " "
1-5 KAH187 20 μg 4.53 15.46μL " ""
  1. Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows:
    1. Label sterile microfuge (1.5 ml) tubes.
    2. Add 570 μL Opti-MEM to each 20 μL DNA sample.
    3. Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
    4. Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.
  2. Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture drop-wise to each appropriate well of cells.
  3. Incubate cells at 37°C in a CO2 incubator
  4. (Optional) To reduce toxicity, after 5-6 hours wash cells once with warm 1xPBS to remove excess DNA-Lipo complexes. Add fresh antibiotic-free growth medium.

Transgene expression should be detectable after 18 hours.




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