User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/08/02: Difference between revisions

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=Date=
=08/02/2013=


'''List title'''
==Procedure==
# List items
# '''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.
# Transfer 5 mL of 100% FBS into a 15 mL conical tube (one per frozen cell vial).
# Retrieve the frozen cell vial from the -150°C freezer.
# Quick-thaw the frozen vial in the 37°C bead bath just until the last of the ice in the tube is thawed. Do this for no more than 2 minutes. '''It took 4 minutes to thaw'''
# 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).
# Get a new '''T-25 flask''' and label it with the label it with the cell line name, your initials, the date, "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.
# Flick the tube to gently resupend the pellet in the ~100 uL FBS.
# Stand the t-25 flask up and remove the cap.
# Add 4 mL of growth medium to the cells. Mix by gently pipetting up and down (about 3 times).
# Transfer the cells + medium into the T-25 flask.
# Lay the flask flat and push it back-and-forth and side-to-side to spread the cells evenly. Do not swirl it in a circular motion.
# Place the flask into the 37°C incubator. The cells should adhere to the growth surface overnight.
# Discard all plastics, etc. that have come into contact with growth medium and cells as biohazard waste.
 
 
Follow-up expansion: After the cells have become 100% confluent in the T-25 flask, aspirate off the growth medium, wash the cells with 2 mL 1xPBS, trypsinize them with 0.5 mL of Trypsin buffer and transfer the entire culture into a T-75 flask with 10 mL total growth medium (10% FBS, 1% pen-strep). After the t-75 is 100% confluent, you can make new frozen stocks or passage the cells at 1:10 (or 1:5 if they are slow-growing).

Revision as of 17:15, 2 August 2013

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08/02/2013

Procedure

  1. Pre-warm the growth medium and 100% FBS (NOT the frozen vial of cells) to 37°C in the bead bath.
  2. 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.
  3. Transfer 5 mL of 100% FBS into a 15 mL conical tube (one per frozen cell vial).
  4. Retrieve the frozen cell vial from the -150°C freezer.
  5. Quick-thaw the frozen vial in the 37°C bead bath just until the last of the ice in the tube is thawed. Do this for no more than 2 minutes. It took 4 minutes to thaw
  6. Use a 5 mL pipette to transfer the 1 mL of thawed cells into the 5 mL of FBS. Do not mix by pipetting.
  7. 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).
  8. Get a new T-25 flask and label it with the label it with the cell line name, your initials, the date, "thawed" to indicate that this is a new culture.
  9. 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.
  10. Flick the tube to gently resupend the pellet in the ~100 uL FBS.
  11. Stand the t-25 flask up and remove the cap.
  12. Add 4 mL of growth medium to the cells. Mix by gently pipetting up and down (about 3 times).
  13. Transfer the cells + medium into the T-25 flask.
  14. Lay the flask flat and push it back-and-forth and side-to-side to spread the cells evenly. Do not swirl it in a circular motion.
  15. Place the flask into the 37°C incubator. The cells should adhere to the growth surface overnight.
  16. Discard all plastics, etc. that have come into contact with growth medium and cells as biohazard waste.


Follow-up expansion: After the cells have become 100% confluent in the T-25 flask, aspirate off the growth medium, wash the cells with 2 mL 1xPBS, trypsinize them with 0.5 mL of Trypsin buffer and transfer the entire culture into a T-75 flask with 10 mL total growth medium (10% FBS, 1% pen-strep). After the t-75 is 100% confluent, you can make new frozen stocks or passage the cells at 1:10 (or 1:5 if they are slow-growing).

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