From 2010.igem.org
(Difference between revisions)
|
|
| Line 1: |
Line 1: |
| - | {{Caltech_iGEM_10|
| |
| - | Content=
| |
| - | __NOTOC__
| |
| - | ==Making Competent Cells==
| |
| - | ===Materials===
| |
| - | * Desired cells
| |
| - | * 1-2L ice cold autoclaved water
| |
| - | * 1L 2YT media
| |
| - | * 2x Erlenmeyer flasks, autoclaved
| |
| - | * Sterile centrifuge tubes large enough to hold 0.2-1L of culture
| |
| | | | |
| - | ===Procedure===
| |
| - | NOTE: The cells must be kept as close to 0°C as possible after they are chilled. All utensils must be completely sterile, as no antibiotics are added to the competent cells, and contamination is a serious danger.
| |
| - | # Incubate two 5mL overnight cultures of the desired cells in 2YT media at 37°C.
| |
| - | # Use the cultures to inoculate two 100-500mL cultures of 2YT in autoclaved Erlenmeyer flasks. Incubate for 2-4 hours.
| |
| - | # Transfer to centrifuge tubes (balanced), chill on ice for 20-30 minutes, and centrifuge at 4000xG for 10 minutes.
| |
| - | # Decant supernatant. Resuspend pellet in an equal volume of ice-cold autoclaved water. Centrifuge again.
| |
| - | # Decant, resuspend pellet in a half-volume of ice-cold water. Centrifuge.
| |
| - | # Decant, resuspend pellet in 1-5mL ice-cold 10% glycerol. Centrifuge.
| |
| - | # Decant, resuspend in 0.5-1mL 10% glycerol.
| |
| - | # Flash-freeze 50μL aliquots in dry ice/ethanol and store at -80°C.
| |
| - | # To be sure that the process was successful, perform positive and negative transformation controls using your new cells.
| |
| - | }}
| |
Latest revision as of 23:42, 14 October 2010
"
