Competent Cells Storage

Competent cells need to be stored at 80 c.

Competent cells storage.

Puc19 and calculate the transformation efficiency of the competent cells. Based on a protocol from kathleen mcginness annotated by josh michener barry canton. One step preparation of competent escherichia coli. Coli cells ideal for construction of gene banks or generation of cdna libraries using plasmid derived vectors.

Most labs store bacteria in 15 25 glycerol. Thermo scientific dh5 competent cells are high efficiency chemically competent e. A typical 200ml bacterial culture produces 20ml of competent cells enough for 800 transformations when using 25ul competent cells per reaction. If the transformation efficiency is low make a new batch of competent cells.

Original protocol published by chung et al. Competent cells supplied in 200µl aliquots are provided with 3ng of competent cells control dna for use as a positive control. Do not refreeze thawed unused aliquots. Chung ct niemela sl and miller rh.

Cells may still be viable but will not transform well. In the morning when you retrieve your liquid bacterial culture take 500 μl of culture to make your glycerol stock before you begin your plasmid mini prep. The optimal concentration of long term glycerol storage is unknown. Cells are made competent by a process that uses calcium chloride and heat shock and cells that are undergoing very rapid growth are made competent more easily than cells in other stages of growth.

Follow the manufacturer s specific transformation protocol. Typically 100µl of competent cells are required for a standard transformation. If using chemically competent cells the incorrect heat shock protocol was used. Cells are not competent transform a plasmid e g.

You can prepare the glycerol stock the same time you prepare your plasmid dna. This means that storing at 20 c can dramatically impede the transformation efficiency. Thaw on ice when ready for use. The competent cells are then aliquoted and stored at 80 o c until we are ready to perform transformations.

Competent cells are those that have had their cell walls altered to render it easier to incorporate foreign dna into their interiors. After just 24 hours of storage at 20 c cells can lose up to 90 of the transformation efficiency. The 80dlaczδm15 marker provides complementation of the galactosidase gene from puc or similar vect. After the final wash suspend the cell pellet in a competent cell solution and aliquot about 10 8 cells per tube each transformation reaction will use these number of cells.

A test reaction with a control plasmid such as puc19 can be performed to determine transformation efficiency of cells stored in this way. Freeze tubes at 80 c more details on storage below. This will give you a stash of competent yeast cells to use in future experiments. Storage of competent cells at temperatures warmer than 70 degrees c even for a brief period will significantly decrease transformation efficiency.