Haynes Lab:Notebook/Jan/2015/04/22: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(Autocreate 2015/04/22 Entry for Haynes_Lab:Notebook/Jan)
 
Line 6: Line 6:
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
==Entry title==
==Transfection of Split U2OS Cells from 4/21/15 with BD004/BD006==
* Insert content here...
* 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 14:46, 22 April 2015

Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Transfection of Split U2OS Cells from 4/21/15 with BD004/BD006

  1. Seed cultures in 6-well plates at ~2.5x105 so that cells will be ~5.0x105 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:

  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. Remove the 6-well plate containing your cultures from the incubator and place it in the biological safety cabinet.
  6. 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.
  7. Add 4mL of warm antibiotic-free growth medium to each well.
  8. Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture drop-wise to each appropriate well of cells.
  9. Incubate cells at 37°C in a CO2 incubator
  10. (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.




Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/Jan/2015/04/22&oldid=881906"