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The document describes procedures for preparing competent E. coli cells and performing bacterial transformation. It involves growing E. coli cells, making them competent through treatment with CaCl2 and glycerol, and introducing plasmid DNA through heat shock. Transformed cells are selected on antibiotic plates and checked for efficiency of transformation.

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Demetrius Daniel
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59 views6 pages

8 and 9

The document describes procedures for preparing competent E. coli cells and performing bacterial transformation. It involves growing E. coli cells, making them competent through treatment with CaCl2 and glycerol, and introducing plasmid DNA through heat shock. Transformed cells are selected on antibiotic plates and checked for efficiency of transformation.

Uploaded by

Demetrius Daniel
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Prac 8

Aim: To study and perform preparation of competent cells


Theory: Competent cells are ready to use bacterial cells that possess more easily
altered cell walls by which foreign DNA can be passed through easily. Most types
of cells cannot take up DNA efficiently unless they have been exposed to special
chemical or electrical treatments to make them competent .
In the CaCl2 method, the competency can be obtained by creating pores in
bacterial cells by suspending them in a solution containing high concentration of
calcium. DNA can then be forced into the Host cell by heat shock treatment at 42C
for the process of transformation.

Materials Required:-
LB broth, Culture plates, Ice cold CaCl2.2H2O (1 M), Ice cold MgCl2 CaCl2
solution, Shaking incubator, Vortex mixer, Centrifuge, Water bath, Inoculation
loop, Microfuge tubes, Polypropylene tubes, Micro pipettes and tips

Reagents Required:
1. 100 mM MgCl2
2. 100 mM CaCl2
3. 85% 100 mM CaCl2, 15% glycerol

Procedure:
Day 1
1. Streak out the E.coli strain on an LBM (LB Miller) plate to isolate
colonies and incubate at 37 degrees C overnight (16-20 hours).
Day 2
1. Use a sterile inoculating loop to collect cells from a single colony and
inoculate 50 ml sterile 1X LBM
2. Grow at 37 degrees C overnight (16-20 hours) in a shaker incubator.
3. Also place 2 flasks of 250 ml 1X LBM in the incubator to equilibrate the
temperature of the medium.
Day 3
1. Add 25 ml of the overnight culture to each 250 ml LBM flask. Place another
flask of 150 ml 1X LBM in the incubator to equilibrate the temperature of
the medium.
2. Grow the cultures to OD600 = 0.1- 0.2.
3. Pellet the cells in chilled autoclaved large centrifuge bottles at 5000 rpm for
10 minutes at 4 degree C.
4. Transport tubes on ice and resuspend on ice in the cold room.
5. Decant supernatant and resuspend the cells in 1/4 original volume (87.5 ml)
ice cold 100 mM MgCl2.
6. Hold on ice for 5 minutes. Spin at 4000 rpm at 4 degrees C.
7. Decant the supernatant and resuspend the cells in 1/20 original volume (17.5
ml) of ice cold 100 mM CaCl2.
8. Hold on ice for 20 minutes. Pellet as above 4000 rpm for 10 minutes at 4
degrees C.
9. Decant the supernatant and resuspend the cell pellet in 1/100 original
volume (3.5 ml) of a solution that is 85% v/v 100 mM CaCl2 and 15% v/v
glycerol (100%).
10.For each culture processed chill approximately 15 labeled eppendorf tubes in
a dry ice-EtOH bath.
11.Pipette 300 ul cells into each tube and place immediately into the dry
ice-EtOH bath. Transfer the frozen competent cell aliquots to -80 degrees C.
12.After the competent cells have been stored for 24 hours check the efficiency
of transformation: Use 1 ng, 10 ng and 100 ng of any ampicillin resistant
plasmid on LBM + Amp plates as per transformation protocol for intact
plasmids.
13.Check the background level by plating 50 ul of cells alone on an LBM +
Amp plate. Expect yields to be approximately 5x107 colonies per ug of
supercoiled DNA.
Precautions:
1. Maintained the required temperature at every step.
2. After CaCl2 treatment bacteria become fragile so do not vortex them.
3. Do not freeze thaw cells many times.
4. Plasmid/cosmid DNA should be considered biohazards and wastes should be
disposed of appropriately.

Prac 9

Aim: To perform bacterial transformation

Theory: ​Transformation- it is the natural process, through which bacterial


populations transfer the genetic material to acquire phenotypic features.
Transformation process allows a bacterium to take up genes from its surrounding
environment; that is transformation involves the direct uptakes of fragments of
DNA by a recipient cell
There are two major parameters involved in efficiently transforming a bacterial
organism. The first is the method used to induce competence for transformation.
The second major parameter is the genetic constitution of the host strain of the
organism being transformed.

Materials Required

LB broth, Ampicillin, LB- Amp plate, Micropipette and tips, Incubator, Water
bath, Microfuge tube, Calcium chloride treated competent cells, spreader

Reagents Required

X Gal: Make a 2% (w/v) stock solution by dissolving X-gal in


dimethylformamide at a concentration of 20 mg/ml solution. The X-gal tube should
wrap with aluminum foil in order to prevent the damage caused by light and store
at -20°C.

IPTG: Make a 20% (w/v, 0.8 M) solution of IPTG by dissolving 2 g of IPTG


with 8 ml of distilled H2O. Prepare the aliquots and store them at -20°C.

Procedure:
1. To transform the CaCl2- treated cells directly, transfer 200 µl of each
suspension of competent cells to a sterile, chilled polypropylene tube using
a chilled micropipette tip.
2. Add DNA (<50ng in a volume of 10 µl) to each tube. Mix the contents of
the tubes by gently swirling. Keep the tubes in ice for 30 minutes.
3. Transfer the tubes to a rack placed in a preheated 42 ºC circulating water
bath. Keep the tubes in the rack for 90 seconds.
4. Transfer the tubes to an ice bath immediately. Allow the cells to chill for 1-2
minutes.
5. Add 800 µl of LB medium to each tube. Incubate the cultures for 45 minutes
in a water bath at 37 ºC .
6. After incubation, add 40 µl of X –Gal and 7 µl of IPTG to the LA-Amp
plate.
7. Add appropriate volume of transformed competent cells into the plate.
8. Spread all the contents uniformly using an L-rod. Keep the plates at room
temperature until the liquid has been absorbed.
9. Invert the plates and keep for incubation at 37 ºC. Transformed colonies will
appear in 12-16 hours of incubation.

Precautions

1. After taking the competent cells from the freezer, it should thaw on ice.
2. To maintain competency bacteria should be kept on ice at all times.
2. There should not be much time lag between adding and spreading EDTA
,Beta-gal and Transformed cells. If so, the components will not spread uniformly in
the plate.
3. Make sure that the samples are uniformly spread into the plate
4. Make sure that the plates with transformed cells should be in an inverted
position while incubating.

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