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
