Materials

• Nalgene cryovials (cat# 366656)
• Nalgene cryoflex tubing (cat# )
• Bunsen Burner
• Pliers

Protocol

• Grow culture until dense (10^6-10^7 cells/ mL or about 2 days).
• Precut cryoflex tubing into 6.5cm lengths.
• Put 900ul cells with 100ul DMSO in cryovials.
• Slide cryovial into pre-cut cryoflex tubing.
• Quickly pass the the covered cryovial through the flame of a bunsen burner and and crimp the ends with a pair of pliers.
• Freeze overnight at 80°C in a rate-controlled (1°/minute) freezer (we use Nalgene’s “Mr. Frosty”).
• The next day, transfer to a liquid nitrogen dewer for long-term storage.