Haynes Lab:Notebook/Jan/2015/04/18: Difference between revisions
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== | ==Transfection of U2OS Cells with BD006 and BD004== | ||
* | * Culture Name: U2OS | ||
* Name of Plasmid: BD006/MV2 and BD004/MV2 | |||
* Benchling Link for Plasmid 1: [https://benchling.com/hayneslab/f/5Clz1JBiCH-mammalian-constructs/seq-W1AJJvtk-chromatinsensor1_mv2/edit BD006/Chromatin Sensor 1] | |||
* Benchling Link for Plasmid 1: [https://benchling.com/hayneslab/f/5Clz1JBiCH-mammalian-constructs/seq-5nN5yYK2-chromatinsensor2_mv2/edit BD004/Chromatin Sensor 2] | |||
* Name of Kit Used: Lipofectamine LTX | |||
* Protocol: [http://openwetware.org/wiki/Haynes:TransfectionPlasmid_Lipo Transfection with Lipofectamine Protocol] | |||
* '''1 day before transfection:'''<br> | |||
# 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. | |||
* '''NOTE:''' if you are using a cell line that does not adhere strongly to the plate (eg, HEK cells), seed the cultures using antibiotic-free medium the day before to avoid having to replace medium the day of transfection. | |||
'''Transfection:'''<br> | |||
# At your bench, bring '''2.0 μg total plasmid DNA''' up to 20 μL total volume with fresh, sterile DNase-free water in fresh, sterile 1.5 mL tubes. Take the DNA samples to the tissue culture room. | |||
# In the tissue culture room, warm antibiotic-free growth medium (e.g., 10% FBS in McCoy’s 5A for, no pen/strep) at '''37°C'''. | |||
# Warm the Lipofectamine LTX vial, PLUS reagent vial, and and a 6 mL aliquot of Opti-MEM I Reduced Serum Medium to '''room temperature''' in the biosafety cabinet. | |||
# Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows: | |||
## Label sterile microfuge (1.5 ml) tubes. | |||
## Add '''570 μL Opti-MEM''' to each 20 μL DNA sample. | |||
## Add '''2.5 μL PLUS reagent''' to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature. | |||
## Add '''7.5 μL Lipofectamine LTX''' to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature. | |||
# Remove the 6-well plate containing your cultures from the incubator and place it in the biological safety cabinet. | |||
# Aspirate off the old antibiotic-containing medium in the wells. '''Do NOT scrape the bottom of the wells, as the cells need to remain adherent to the plate'''. | |||
# Add 4mL of warm antibiotic-free growth medium to each well. | |||
# Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture '''drop-wise''' to each appropriate well of cells. | |||
# Incubate cells at 37°C in a CO<sub>2</sub> incubator | |||
# (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. | |||
Revision as of 11:44, 18 April 2015
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Transfection of U2OS Cells with BD006 and BD004
Transfection:
Transgene expression should be detectable after 18 hours.
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