Skip to main content

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! :)

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

    ReplyDelete

Post a Comment

Popular posts from this blog

Lowry Assay Principle and procedure

Though there are several protein assays available, the most preferred one in many laboratories is "Lowry assay". It is effective in the concentration range of 0.01 mg/ml to 1 mg/ml. And, as an additional info, the paper published describing the procedure and principle of Lowry Assay is the most cited paper in the scientific history. (Feeling like, "Wow! I want to publish one to compete with Oliver.H. Lowry"???  :P) Why Lowry?   Though there are several other protein assays, mostly Lowry assay is used in many laboratories. The reasons for preferring Lowry are: sensitivity of the assay, highly reproducible, cost effective, easy to perform. Biuret assay is generally used for higher protein concentrations like tissue samples but, Lowry for less concentrated samples and hence used in most of the molecular biology laboratories where there will be need for assaying comparatively less concentrated protein samples (in most cases where we attempt to produce enzymes). Oth

Sea Butterfly!

Neih hou!  Don't roll you eyes wondering what it is! "Neih hou" is how you say "hello" in Cantonese. Guess what, I am in Hong Kong and therefore the language Cantonese. I came to Hong Kong this summer as an intern before officially joining as a PhD Student. This is my second experience abroad, far away from home, new language, new culture, I expected me to have a "really" bad cultural shock, but, actually I experienced only 50% of what I expected. For a girl who have used the marina beach only for eating "sundal", bikini in beach was a shock (FEEL FREE TO JUDGE ME!). The first time, I went to the big wave beach here, I was the only one who was totally covered, when everyone was enjoying their Beer, I was slowly sipping my lemon juice (THE ODD ONE OUT!). I realized how beautifully different the world outside is! There is no single standard for right or wrong, it varies in different countries, in different regions around the world. And it

Isolation of monocytes from PBMC (Peripheral Blood Mononuclear Cells) - Principle and protocol

Whenever I'm made to realise that I'm not clear enough or good at something, I try to make myself clear with it. It happened today, during my laboratory examination, I was asked to perform monocyte isolation from a given blood sample, but, unfortunately, I was not very clear with the principle behind it.(but, still I managed to complete the experiment as I know the protocol, but, knowing the principle behind each step of the protocol clearly is very important, isn't it?). But, nothing is wrong in it, I made myself clear with it now. That's good, right? So, let me share with you some basic principle and protocol for isolating monocyte from blood sample. For isolating monocytes, initially we must isolate PBMC (Peripheral bood mono nuclear cells) from the blood sample. Here, let us make few terminologies clear before starting with the principle. Peripheral blood sample  - It is the blood sample obtained from acral areas of body (in general, it is the blood collected