User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/06/06: Difference between revisions
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# 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. | |||
# 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 12:07, 6 June 2013
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06/06/2013
Transfections
BD002 (3:1) = 339 ng/μl BD002 (4:1) = 317 ng/μl PlpE = 192 ng/μl
Transgene expression should be detectable after 18 hours.
Polycomb-ATF negative control lines: colony plates
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