User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/08/02: Difference between revisions
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=Thawing Cells (U2OS Flp-TRX)= | |||
=Procedure= | |||
# '''Pre-warm the growth medium and 100% FBS (NOT the frozen vial of cells) to 37°C in the bead bath'''. | # '''Pre-warm the growth medium and 100% FBS (NOT the frozen vial of cells) to 37°C in the bead bath'''. | ||
# After reagents are warmed, spray bottles down with 70% ethanol and prepare the biosafety hood as for routine work. Do all work in the biosafety hood. | # After reagents are warmed, spray bottles down with 70% ethanol and prepare the biosafety hood as for routine work. Do all work in the biosafety hood. | ||
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# Use a 5 mL pipette to transfer the 1 mL of thawed cells into the 5 mL of FBS. Do '''not''' mix by pipetting. | # Use a 5 mL pipette to transfer the 1 mL of thawed cells into the 5 mL of FBS. Do '''not''' mix by pipetting. | ||
# Spin the cells + FBS at room temperature at 1000 rpm for 3 minutes. If you have just one tube, use a counter balance of equal volume (6 mL water). | # Spin the cells + FBS at room temperature at 1000 rpm for 3 minutes. If you have just one tube, use a counter balance of equal volume (6 mL water). | ||
# Get a new '''T-25 flask''' and label it with the label it with | # Get a new '''T-25 flask''' and label it with the label it with '''U2OS FlpE-TRX, BD, 08/03/2013, "thawed" '''to indicate that this is a new culture. | ||
# After the cells have pelleted, bring them back to the biosafety cabinet and aspirate off the FBS, but leave about 100 uL of FBS covering the pellet. | # After the cells have pelleted, bring them back to the biosafety cabinet and aspirate off the FBS, but leave about 100 uL of FBS covering the pellet. | ||
# Flick the tube to gently resupend the pellet in the ~100 uL FBS. | # Flick the tube to gently resupend the pellet in the ~100 uL FBS. |
Revision as of 17:18, 2 August 2013
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08/02/2013Thawing Cells (U2OS Flp-TRX)Procedure
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