Preparation of electrocompetent cells

About me

I am a girl who is carrying lot more dreams in her eyes.Rarely i speak in words. I like to imagine lot more things. First picture or image come into my mind and then i may try to express it in words. I am always curious to know new information and techniques. Biotechnology is my passion and cooking is my hobby.when hobby and passion meet together it will lead to biorecipe.

Actually doing experiments in the lab and doing cooking in the kitchen involves similar biological and chemical reactions.So i combine both the thing and i am introducing biorecipe. You will be doing experiments in step by step manner under some ambient conditions and finally your recipe will be ready, for example in plasmid isolation finally you will get a plasmid DNA, that is your final recipe ya its biorecipe. I am here to post some basic techniques in biotechnology field which includes genetic engineering,molecular biology, immunology etc.

It will help aspiring students to explore biotechnology. As einstein said, “imagination is more important than knowledge”, picturise what you are reading. So readers are you ready to travel with me.

Preparation of electrocompetent cells

What are electrocompetent cells?

Literally competent means efficient. We make cells to become competent inorder to tranform our desired plasmid into E.coli efficiently.Naturally bacteria can take up exogenous DNA in three different ways: conjugation, transformation and transduction. In conjugation DNA is transferred via pilus (Fig 1.1) which requires cell- cell contact, transduction is mediated by bacteriophages to transfer DNA from one cell to another. But transformation involves direct uptake of DNA. Naturally series of competence proteins are produced inside the cells to facilitate the transfer the DNA from one cell to another. To make cells more efficient for transformation, cell walls are altered in such a way that cells are chemically competent or electrocompetent.

Fig 1.1

Applications of electrocompetent cells

Efficient uptake of plasmid DNA by transformation of competent E.coli is a crucial step in molecular cloning and is required in order to perform numerous downstream applications such as recombinant protein expression and mutagenesis.

Readers be ready to do biorecipe 1 electrocompetent cell preparation.

Note: imagine you are in the lab while you are reading this post.

Aim

To prepare the competent cells of E.coli DH5α strain

Principle

In earlier days, it was found that E.coli cells soaked in ice cold solution were more efficient in receiving foreign DNA than normal cells. During competent cell preparation, it is essential to extensively wash cells to remove residual culture medium and  reduce the ionic strength of the cell suspension while ensuring its high resistance and very low conductivity. It is necessary to avoid the interference of salts that could reduce electrotransformation efficiency. 15% ice cold glycerol washes of harvested cells reduce the ionic strength of the cell suspension and removes the residual culture medium and increase the density of cell to settle down.

Materials required to prepare biorecipe (competent cell)

1. Strain      E.coli DH5α, BL21 (DE3) strains

2. Reagents   LB components, 500 mL of sterile 15% glycerol, 1 L of sterile distilled water, ethanol for cleaning, dry ice

3. Apparatus  Pipettes, laminar flow hood, autoclave, spectrophotometer, pHmeter, incubator, centrifuge, shaker.

LB media preparation

LB broth Add 4 gm of yeast, 8 gm of tryptone, 8 gm of NaCl to 800 mL of distilled water and the pH should be adjusted to 7.3

LB agar Take 200 mL of LB broth in a 250 mL conical flask and add 3 gm of agar.

The culture tubes, LB agar, LB broth, petriplates, eppendorfs, 15% glycerol and 1 L of sterile double distilled water (DD) should be autoclaved for 20 minutes at 120˚C

Media inoculation

Add 3 mL of LB broth to 10 test tubes each using micropipette.

Take a single colony from culture of DH5α or BL21 (DE3) strain and immerse it in a culture tube containing LB broth. Place all the test tubes in the shaker and incubate it for overnight at 37˚C.

Streaking for storage

Take loopful of culture from the tube using sterile inoculation loop and streak the LB agar plates. Incubate at 37˚C for overnight and then place it in the refrigerator.

Procedure

1. Take overnight culture and check O.D value at 600 nm

2. Inorder to attain an initial OD of 0.05 at 600 nm subculture 250 mL of LB broth with overnight culture. You have to calculate how much volume of overnight culture should be added to 250 mL of LB broth to attain OD of 0.05 according to your initial OD value

For example : if your OD value for overnight culture is 1.97 then required volume will be

0.05*250/1.97 (Dilute your sample prior to check OD and actual OD= Obtained OD*dilution factor)

3. The OD at 600 after 2 hours preferable to be between 0.6-1.0

4. Keep 15% glycerol, DD, and falcon tubes in buckets of ice for prechilling

5. Transfer 125 mL of culture into two 250 mL centrifuge tubes and centrifuge at 6000 rpm for 7 minutes each at 4˚C

6. The culture should be prechilled before centrifuging

7. Decant the supernatant without disturbing the pellet

8. Resuspend the pellet in 50 mL of cold water (sterile) gently and centrifuge it for 6000 rpm for 4 minutes

9. Discard the supernatant and repeat above step twice

10. Wash your pellet with 15% glycerol (50 mL) and spin at 4000 rpm for 5 minutes.Discard the supernatant

11. Resuspend the pellet in 0.5 mL of 15% glycerol

12. Freeze the eppendorfs containing aliquots of bacteria in dry ice/ethanol bath

13. Transfer it into ultra freezer (-80˚C) and store it for further use.

14. Now the biorecipe is ready for transformation or any other purpose.

Precaution

1. All the inoculation procedure should be done under the hood

2. The hood should be cleaned with alcohol

3. The loops and mouth of the test tubes and flasks must be flamed thoroughly before and after use

4. Conical flasks and petriplates with culture should not be opened anywhere except in the hood

5. It is essential to maintain everything in the ice cold

6. A loopful of competent cell should be streaked on LB ampicillin plate and incubated to evaluate the quality of competent cells.

NOTE: Cells should be devoid of plasmid DNA unless we can’t use it for transformation. To check this, competent cell should be cultured on LB ampicillin plate or any antibiotic plate accordingly.

Example: if you are handling plasmid DNA containing Amp resistant genes in your lab most predominantly, then you have to culture your competent cells on LB ampicillin plate