Helpful tips

What are top 10 cells?

by Chase Collins

What are top 10 cells?

TOP10 cells are lacIq- (minus). They do not have the lacIq gene and therefore do not produce the lacIq repressor protein. lacIq is most commonly found on an F’ episome, and therefore is present in TOP10F’, JM101, JM109, and NM522 strains.

Do competent cells expire?

If you are using chemically competent cells they should be OK for a few years if properly stored. Budak- these cells will work fine. Companies won’t guarantee the performance of competent cells past their use-by date, but they don’t expire in the sense of suddenly failing to work altogether.

How do you choose competent cells?

Before selecting competent cells, you need to consider the expected results of your transformation. For protein expression, you will need a different strain of competent cells than what you would use for regular cloning.

How do you make a Top 10 competent cell?

Preparing seed stocks

  1. Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C.
  2. Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C.
  3. Add glycerol to 15%
  4. Aliquot 1 ml samples to Nunc cryotubes.
  5. Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes.

What is BL21?

BL21(DE3) is an E. coli B strain and does not contain the lon protease. It is also deficient in the outer membrane protease OmpT. The lack of these two key proteases reduces degradation of heterologous proteins expressed in the cells.

Can I store competent cells at?

Competent cells should be stored at -80°C. Storage at -20°C will result in a significant decrease in transformation efficiency (TE).

How do competent cells grow?

How to Make Chemically-Competent Cells. Take the strain of E. coli you wish to make chemically-competent from either a glycerol stock or a freshly-streaked agar plate and inoculate it into a flask containing approximately 50 mL of RB. Let it grow overnight.

What are the types of competent cells?

There are two types of artificially competent cells: chemically competent and electrocompetent.