Storing and using competent cells

Storing Competent Cells:

The competent cells used in the ABE labs need to be stored at -80°C to stay competent. To facilitate this, we deliver your cells as close to your Lab 5/5a date as possible. 

Your cells will be delivered in a foam cooler, full of dry ice pellets, with the cells in a small zip bag underneath the dry ice. Store the entire cooler, with the lid tightly closed, in a regular freezer as soon as possible. Depending on conditions, the dry ice should last at least 24 hours if stored properly. As long as there is dry ice, your cells will stay competent. If needed, you can add to the dry ice supply (many grocery and liquor stores sell dry ice, just call in advance to check).  

If you are receiving the cells more than 24 hours before your planned date for Lab 5/5a, you will also receive an isofreeze rack. This rack can store the competent cells up to one week in a regular freezer and maintain their competency. If you receive an isofreeze rack, immediately remove it from the cooler you received it in and put the isofreeze rack and lid in your freezer. Make sure there is plenty of room for air to circulate around the isofreeze rack. Store the competent cells as noted above until the isofreeze rack has completely refrozen (ideally overnight). Then remove the competent cells from the dry ice, being careful to only handle the tubes by the lid and not touch the bottom, and put them in the isofreeze rack in the freezer. 
 

Using Competent Cells:

The competent cells are very sensitive to temperature. They must be defrosted on wet, crushed ice just before use. There are 500µL of competent cells per tube, and each lab group will need 100µL of competent cells. ONLY defrost enough competent cells for the class you are teaching (e.g. if 1st period has 5 lab groups defrost only 1 tube). They take about 15 minutes to defrost. 

Do NOT pre-aliquot the competent cells for students. Have students pre-chill their P- and P+ tubes on ice for at least a minute. Have students bring their cup of ice with the pre-chilled tubes in it to you and aliquot the competent cells directly into their tubes (50µL per tube). Be careful not to touch the bottom of any of  the tubes, as this will warm the competent cells decreasing their competency (i.e. leads to poor lab results!). Try to aliquot the tubes holding them just above the ice so that you can cap them and put them directly back on ice. Do not leave the tubes uncapped, as this can introduce contamination and splashes of water can get into the tube from the melting ice.

Be sure students pay careful attention to temperature and timing! This will lead to the best results for this lab.