Sunday, July 22, 2012

Electrocompetent Cells of DH5 alpha

Electro competent cells are prepared for DNA transformation. Usually the cell wall of bacteria are very thick and hence, a foreign plasmid DNA can't penetrate into it, very easily. So, we prepare electrocompetent cells, which have reduced cell wall thickness or pores over the cell wall.

So, due to this pores, DNA can easily enter the cell and thus transformation is made easy. In our lab, we did prepare, electrocompetent cells of E.coli DH5 alpha. The procedure for preparing electrocompetent cells is available in various sites. So, am not going to discuss the procedure. If you wanna know it, kindly mail me, I will send you.

Okay, following the procedure, you can prepare electrocompetent cells, but, how to ensure that you had prepared a non contaminated one? Generally, Plating is done.

Just streaking, a LB plate and an Ampicillin LB plate with the cells. You must observe, growth in LB plate and no growth with the Ampicillin plate. If so, then pat your back, you did it.

What's the basic behind this plating? Why an Ampicillin plate?

Generally, E.coli DH5 alpha is not resistant against ampicillin. It will show resistance only after you transform it with a plasmid. (Plasmid gives resistance)

So, If you have contamination with other microbial cells, you will find growth in LB Ampicillin plate too.

:) Got it? Any queries? Mistakes? Comment then! :)

Happy learning! :)

2 comments:

  1. Resistence depends on the plasmid u are using...for pET-33b Kanamycin antibiotic is used...During the addition of antibiotic if the agar is very hot then antibiotic will fail to work

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