Difference between revisions of "User:Brendan F. Fries/Notebook/G-Bioscience Extraction of Genomic DNA from LUC-14 Cells"

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==LUC-14 Cells grown in DMEM media 2/19/2014==
 
* LUC-14 cells were passesed into 6-well plate and grown for 48 hours to ~90% confluency to obtain approximately 1*10^6 cells. This was done in accordance with HEK-293 cell numbers per tissue plate at confluency per [http://www.eppendorf.com/content/1/1/doc/Eppendorf_Tissue_Culture_Consumables_cell_line_performance_HEK293.pdf].
 
  
==Genomic DNA extraction 2/21/2014==
 
* Steps done in tissue culture room
 
#2mL PBS added to well
 
#0.5mL Trypsin added
 
#added to 15mL centrifuge tube
 
#4.5 DMEM cell culture medium
 
#centrifuged at 600g for 5 min
 
#pipetted to 1.5ml centrifuge tube
 
* Taken out of culture room
 
#supernatant was disposed of leaving ~20uL residual which was vortexed.
 
#400uL XIT Lysis Buffer added and mixed via vortex.
 
#90uL XIT Protein Precipitation Buffer and tube was inverted 15 times.
 
#centrifuged at 14,000g for 2 min.
 
#precipitate not settled, incubated on ice and centrfiguged again for 5 min. Transferred supernatant to new 1.5mL tube.
 
#400ul isopropanol added and tube inverted 40 times.
 
#centrifuged at 14,000g for 5 min.
 
#centrifuged at 14,000g for 2 more min.
 
#supernatant discarded with pipette and washed with 200uL 70% ethanol by inverting two times.
 
#centrifuged at 14,000g for 2 min.
 
#centrifuged at 14,000g for 2 more min.
 
#discarded supernatant and left to air dry for 15 min.
 
#50uL TE Buffer (pre-warmed to 55 C) and 1uL thawed RNase.
 
#Incubated at 55 C for 1 hour.
 
#Left to rehydrate at room temp overnight.
 
* Genomic DNA concentration measured 2/22/14
 
#  1.788  260/280
 
#  868.163 ng/uL
 
 
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Revision as of 00:53, 26 February 2014

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G-Biosciences XIT Genomic DNA extraction from cultured cells

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