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

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(October 23, 2013)
(October 23, 2013)
Line 13: Line 13:
| Plate-Well || Plasmid || DNA || Volume|| dH2O|| Lipo  || PLUS reagent || <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 || 8.187 + 2.60 μL || 9.22μL|| 2.5μL|| 7.5 μL || 570 μL
+
| 1-2           || KAH126 || 1.5 μg || 23 μL > 20 μL || 0μL|| 2.5μL|| 7.5 μL || 570 μL
|}
|}

Revision as of 12:32, 25 October 2013



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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.



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