User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/06/10

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(Autocreate 2013/06/10 Entry for User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli)
Current revision (16:58, 23 July 2013) (view source)
(Date)
 
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=Date=
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=06/10/13=
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'''List title'''
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# List items
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'''1 day before transfection:'''<br>
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# Seed cultures in 6-well plates at ~2.5x10<suP>5</sup> so that cells will be ~5.0x10<sup>5</sup> at the time of transfection.
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'''Transfections'''<br>
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> U2OS Flp-in T-REx lines (puro/zeo/blast) + FRT-marked mammalian transfection vector (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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# BD002 (3:1) Flp-in T-REx
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#BD002 (4:1) Flp-in T-REx
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BD002 (3:1) = 183.42 ng/μl
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BD002 (3:1) = 317.00 ng/μl
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BD002 (4:1) = 346.61 ng/μl
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KAH187 = 441.5 ng/μl
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FlpE = 192 ng/μl
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{| {{table}} cellspacing="7"  width=700px
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|-
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| Plate-Well || Plasmid || DNA || Volume|| dH2O|| Lipo  || PLUS reagent || <u>Opti-MEM (total)</u>
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|-
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| 1-1            || BD002 (3:1) + FlpE  || 1.5 + 0.5 μg || 8.187 + 2.60 μL || 9.22μL|| 2.5μL|| 7.5 μL || 570 μL
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|-
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| 1-2            || BD002 (4:1) + FlpE || "          || 4.32 + 2.60 μL  || 13.08μL|| "  ||    "      || "
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|-
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| 1-3    || BD002 (4:1) + FlpE || 1.5 + 0.5 μg      || 4.73 + 2.6 μL  || 12.67μL || "    ||  "    || "
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|-
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| 1-4 (-)Ctrl      ||  BD002 (3:1) + --- || 2 μg        || 8.18 + --- μL  || 11.82μL|| "    ||    "    || "
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|-
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|1-5 (-) Ctrl      ||  ---    ||    ---        ||  ---  || 20μL||    "  || "||"
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|-
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|1-6            || KAH187    ||  2 μg        ||  4.53  || 15.46μL||    "  || "||"
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|}
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# Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows:
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## Label sterile microfuge (1.5 ml) tubes.
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## Add '''570 μL Opti-MEM''' to each 20 μL DNA sample.
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## Add '''2.5 μL PLUS reagent''' to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
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## Add '''7.5 μL Lipofectamine LTX''' to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.
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# Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture '''drop-wise''' to each appropriate well of cells.
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# Incubate cells at 37°C in a CO<sub>2</sub> incubator
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# (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.
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Transgene expression should be detectable after 18 hours.
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----
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__NOTOC__

Current revision

PcTF Subcloning in E. coli Main project page
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06/10/13

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) = 183.42 ng/μl BD002 (3:1) = 317.00 ng/μl BD002 (4:1) = 346.61 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 8.187 + 2.60 μL 9.22μL 2.5μL 7.5 μL 570 μL
1-2 BD002 (4:1) + FlpE " 4.32 + 2.60 μL 13.08μL " " "
1-3 BD002 (4:1) + FlpE 1.5 + 0.5 μg 4.73 + 2.6 μL 12.67μL " " "
1-4 (-)Ctrl BD002 (3:1) + --- 2 μg 8.18 + --- μL 11.82μL " " "
1-5 (-) Ctrl --- --- --- 20μL " ""
1-6 KAH187 2 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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