- 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.
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