User:Brendan F. Fries/Notebook/G-Bioscience Extraction of Genomic DNA from LUC-14 Cells/2014/02/21: Difference between revisions

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==Entry title==
==Genomic DNA extraction 2/21/2014==
* Insert content here...
* 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.




Revision as of 00:42, 26 February 2014

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Genomic DNA extraction 2/21/2014

  • Steps done in tissue culture room
  1. 2mL PBS added to well
  2. 0.5mL Trypsin added
  3. added to 15mL centrifuge tube
  4. 4.5 DMEM cell culture medium
  5. centrifuged at 600g for 5 min
  6. pipetted to 1.5ml centrifuge tube
  • Taken out of culture room
  1. supernatant was disposed of leaving ~20uL residual which was vortexed.
  2. 400uL XIT Lysis Buffer added and mixed via vortex.
  3. 90uL XIT Protein Precipitation Buffer and tube was inverted 15 times.
  4. centrifuged at 14,000g for 2 min.
  5. precipitate not settled, incubated on ice and centrfiguged again for 5 min. Transferred supernatant to new 1.5mL tube.
  6. 400ul isopropanol added and tube inverted 40 times.
  7. centrifuged at 14,000g for 5 min.
  8. centrifuged at 14,000g for 2 more min.
  9. supernatant discarded with pipette and washed with 200uL 70% ethanol by inverting two times.
  10. centrifuged at 14,000g for 2 min.
  11. centrifuged at 14,000g for 2 more min.
  12. discarded supernatant and left to air dry for 15 min.
  13. 50uL TE Buffer (pre-warmed to 55 C) and 1uL thawed RNase.
  14. Incubated at 55 C for 1 hour.
  15. Left to rehydrate at room temp overnight.



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