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

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* Place short description of project or notes regarding this project
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==LUC-14 Cells grown in DMEM media 2/19/2014==
 
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* 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].
 
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==Genomic DNA extraction 2/21/2014==
 
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* Steps done in tissue culture room
 
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#2mL PBS added to well
 
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#0.5mL Trypsin added
 
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#added to 15mL centrifuge tube
 
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#4.5 DMEM cell culture medium
 
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#centrifuged at 600g for 5 min
 
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#pipetted to 1.5ml centrifuge tube
 
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* Taken out of culture room
 
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#supernatant was disposed of leaving ~20uL residual which was vortexed.
 
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#400uL XIT Lysis Buffer added and mixed via vortex.
 
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#90uL XIT Protein Precipitation Buffer and tube was inverted 15 times.
 
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#centrifuged at 14,000g for 2 min.
 
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#precipitate not settled, incubated on ice and centrfiguged again for 5 min. Transferred supernatant to new 1.5mL tube.
 
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#400ul isopropanol added and tube inverted 40 times.
 
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#centrifuged at 14,000g for 5 min.
 
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#centrifuged at 14,000g for 2 more min.
 
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#supernatant discarded with pipette and washed with 200uL 70% ethanol by inverting two times.
 
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#centrifuged at 14,000g for 2 min.
 
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#centrifuged at 14,000g for 2 more min.
 
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#discarded supernatant and left to air dry for 15 min.
 
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#50uL TE Buffer (pre-warmed to 55 C) and 1uL thawed RNase.
 
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#Incubated at 55 C for 1 hour.
 
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#Left to rehydrate at room temp overnight.
 
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* Genomic DNA concentration measured 2/22/14
 
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#  1.788  260/280
 
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#  868.163 ng/uL
 
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Revision as of 02:53, 26 February 2014

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G-Biosciences XIT Genomic DNA extraction from cultured cells

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