Haynes Lab:Notebook/Engineering PC-TFs/2013/10/23

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(Autocreate 2013/10/23 Entry for Haynes_Lab:Notebook/Engineering_PC-TFs)
Current revision (12:35, 25 October 2013) (view source)
(October 23, 2013)
 
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==Summary==
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==October 23, 2013==
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*  
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'''Transfection of KAH126'''
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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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| 1-2            || KAH126  || 1.5 μg || 23 μL > 20 μL || 0μL|| 2.5μL|| 7.5 μL || 570 μL
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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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'''Cell Culture of KAH126'''
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''Grow liquid culture of KAH126''
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*Label 15 ml sterile culture tubes. Fill each tube with 2 ml LB growth medium with Amp.
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*Use sterile pipette tip to touch bacterial streak and put the tip into LB medium.
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*Grow the cultures for 10-12 hrs.
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October 23, 2013

Transfection of KAH126

Plate-Well Plasmid DNA Volume dH2O Lipo PLUS reagent Opti-MEM (total)
1-2 KAH126 1.5 μg 23 μL > 20 μL 0μL 2.5μL 7.5 μL 570 μL
  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.

Cell Culture of KAH126

Grow liquid culture of KAH126

  • Label 15 ml sterile culture tubes. Fill each tube with 2 ml LB growth medium with Amp.
  • Use sterile pipette tip to touch bacterial streak and put the tip into LB medium.
  • Grow the cultures for 10-12 hrs.



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