PREPARATION AND TRANSFORMATION OF COMPETENT E.
coli
USING THE HEAT SHOCK METHOD
OBJECTIVE: To prepare competent E. coli using Calcium Chloride and transform them
with the plasmid vector pBR322. You will also calculate the Transformation transformation
Efficiencyefficiency.
THEORY:
Give some theory about natural transformation and artificial transformation.
Need for making the cells competent.
The process of insertion and expression of foreign genetic material into a bacterial cell is
known as Ttrransformation. This can be done using two ways:
1. Heat Shock Method
Formatted: Bullets and Numbering
2. Electroporation Method
In the case of the Heat Shock Method, competent cells are incubated with DNA on ice then
subjected to a sudden increase in temperature to 42C for a brief period of 45-120 sec.
Comment [MU1]: Check this value
Following the heat shock, the cells are quickly cooled. At the cellular level, this heat shock
involves destabilization of the membrane of the cell that leads to the uptake of the
surrounding liquid that includes the foreign nucleic acid.
In order for the process of transformation to be successfully efficient the bacterial host cells
must be made competent, referring to their state of being able to take up exogenous DNA
from the environment. Cells can be made competent by chilling them on ice and washing
them repeatedly with CaCl2.
Ca
2+
Comment [MU2]: This paragraph should go
earlier
acts as a shield between the negatively charged lipids of the cellular membranes and
negatively charged plasmid DNA. The absence of such a positive ion would have otherwise
resulted in like-charge repulsion between the bacterial cell membrane and DNA thereby
decreasing efficiency of uptake of the plasmid. In this protocol, an additional cation, Mg 2+ is
also used to increase efficiency.
The plasmid used in this experiment is pBR322. It is a vector which has selectable marker
genes that encode for proteins offering resistance to both Ampicillin and Tetracycline. Thus
cells that have successfully been transformed with pBR322 will be able to grow in media
with Amp and Tet. This resistance conferred onto transformed cells is used as a method of
selection.
Comment [MU3]: Mention the size
MATERIALS REQUIRED:
Glassware: Culture Tubes, Conical Flasks, Beakers, Measuring Cylinders, Spreader, Petriplates
Apparatus: Shaker Incubator, Spectrophotometer, Laminar, Centrifuge, Water bath,
Micropipettes and Microtips, 1.5 ml Microfuge tubes, Polypropylene Oakridge tubes,
Crushed Ice, Blotting Paper, Toothpicks or Inoculation Loop
Chemicals Required:
CHEMICAL
STOCK CONC.
WORKING CONC.
MOL. WT.
CaCl2
1 M (50ml)
100mM and 20mM
111 g/l
MgCl2
1 M (50ml)
80mM
203.31 g/l
Tris-Cl
1 M (250ml)
121 g/l
EDTA
0.5 M (100ml)
186 g/l
Ampicillin
100 mg/ml (1000X)
1X
Carbenicillin
50 mg/ml (1000X)
1X
Tetracyclin
50 mg/ml (1000X)
1X
Glycerol
100%
15%
TE BUFFER: (For 100 ml):
o 1 ml of 1 M Tris-Cl
o 20ul of 0.5 M EDTA
SOC MEDIA: (For 100ml):
o 2.0g Tryptone
o 0.5g Yeast Extract
o 0.5g NaCl
o 0.36g Glucose
100 ml of MgCl2 (80mM) - CaCl2 (20mM) solution
300 ml LB Agar (For 8+1 plates)
100 ml for LB Broth + 10 ml LB Broth in a culture tube
Culture used:E. coli strain DH5.
DNA Plasmid: pBR322 (Stock Concentration: 330ng/)
Formatted: Bullets and Numbering
Plasmid Dilution:Working Concentration: 1 ng/ ul.
This can be achieved by serial dilution.
330 ng/ul 10 ng/ul 1 ng/ul
TRANSFORMATION EFFICIENCY:(CFU/g plasmid)
Number of colonies x 1000
% cells plated x Amount of DNA added (ng)
EXPERIMENTAL SET UP:
In order to check the success of transformation, you will maintain 2 sets of plates, TEST and
CONTROL.
PLATING SCHEME FOR TEST:(% Check up)
1000l E.coli + pBR322
LB only ( )
LB + Amp +Tet ( )
LB+Amp ( )
LB+Amp+Tet ( )
LB+Tet ( )
PLATING SCHEME FOR CONTROL:
1000l E.coli + TE Buffer
LB only ( )
Formatted: Font: Italic
LB+Tet ( )
LB+Amp ( )
PREPARATORY STEPS:
1. All working concentrations of reagents to be used for this experiment must be
prepared from the Stock Solutions.
2. As per the plating scheme illustrated above, prepare LB Agar required for pouring 8
plates. Also account for 1 plate which you will use to subculture E. coli for this
experiment.
3. Autoclave all glassware, reagents, LB Broth and LB Agar.
Formatted: Bullets and Numbering
PREPARATION OF COMPETENT CELLS
1. Pick a single, fresh bacterial colony (2-3 mm) in diameter from the subcultured E.coli
DH5 plate that has been incubated for 16-20 hours at 37C. Transfer the colony into
Comment [R4]: Sir, I havent mentioned the
culturing of DH5 in culture tubes from which we
do streak plating. Is it required? Directly started w
pick from plate.
Formatted: Bullets and Numbering
Formatted: Font: Italic
100ml of LB Broth in a 500ml flask. Alternatively, you may use 50-100l of logphase starter culture for inoculation into broth. Incubate the culture for 3 hours at
37C in a shaker incubator at 200 RPM.
For efficient transformation, it is essential that the number of viable cells not exceed
108 which corresponds to an OD of 0.4. (Ensure that the OD DOES NOT cross this
value)
2. Transfer the bacterial cells to sterile, disposable, ice-cold 50ml polypropylene tubes or
Formatted: Bullets and Numbering
Oakridge tubes. Cool the cultures to 0C by storing the tubes on ice for 10 mins.
3. Recover the cells by centrifuging at 2700g for 10 min at 4C .
You should observe a small pellet
The centrifuge should be prechilled to 4C before use.
4. Decant the medium from the cell pellets. Stand the tubes in the inverted position on
Formatted: Bullets and Numbering
blotting paper for 1 min to allow the last traces of media to drain away.
It is preferred that this step be done in the laminar to avoid contamination from AmpR
Bacteria in the surroundings
5. Resuspend each pellet by swirling or gentle vortexing in 30ml of ice-cold MgCl2-
Formatted: Bullets and Numbering
CaCl2 solution (80mM MgCl2, 20mM CaCl2)
You will observe that the pellet becomes fluffy
6. Recover the cells by centrifugation at 2700g for 10 mins at 4C
Formatted: Bullets and Numbering
7. Decant the medium from the cell pellets. Stand the tubes in the inverted position on
blotting paper for 1 min to allow the last traces of media to drain away.
8. Resuspend the pellet by swirling or gentle vortexing in 2 ml of ice-cold 0.1 M CaCl2
Formatted: Indent: Left: 0.25", Space Afte
0 pt
for each 50 ml of original culture.
9. At this point, you have 2 options
a. Use the cells directly for transformation
b. Dispense into aliquots of 200l as 15 % Glycerol Stock in prechilled 1.5 ml
eppendorfs using pre-chilled tips and freeze at -70C.
Formatted: Indent: Left: 0.25"
Formatted: Bullets and Numbering
TRANSFORMATION
Start numbering again
8. Include all positive and negative controls. In this experiment, you are transforming
Formatted: Bullets and Numbering
competent E.coli with pBR322 which has Amp R and TetR. Please refer to the
EXPERIMENTAL SETUP for details on the plating scheme.
9. Take 8 tubes each containing aliquots of 200ul of competent cellsprepared and add
the plasmid DNA (No more than 50ng in a volume of 10l or less) to the Test tubes
only.To the Control tubesadd an equal amount of TE buffer instead. Mix the
contents of the tubes by swirling gently. Store the tubes on ice for 30 mins.
10. Transfer the tubes to a rack placed in a preheated 42C circulating water bath. Store
the tubes in the rack for exactly 90 seconds. Do not shake the tubes.
Heat shock is a crucial step. It is very important that the cells be raised to exactly the
right temperature at the correct rate.
11. Rapidly transfer the tubes to an ice-bath. Allow the cells to chill for 1-2 mins.
Formatted: Bullets and Numbering
12. Add 800l of SOC Medium to each tube (thus making the contents of each tube 1000
l). Incubate the cultures for 45 mins in a water bath set at 37C to allow the bacteria
to recover and to express the antibiotic resistance marker encoded by pBR322.
13. Transfer the appropriate volume of transformed competent cells using spread plate
technique onto the LB Agar plates which have the appropriate antibiotic.
To plate rest, spin down the remaining contents of the tube, discard the supernatant and
plate the now concentrated remainder.
14. Invert the plates and incubate at 37C. Transformed colonies should appear in 12-16
hours. Store the plates at 4C after colonies appear.
EXPECTED RESULTS:
WITH pBR322
LB ONLY
LB + AMP
LB+ TET
LB+AMP+TET
Formatted: Bullets and Numbering
NO DNA (control)
+ PRESENCE OF COLONIES
ABSENCE OF COLONIES
PRECAUTIONS:
1. Always take a sample LB and store it for blanking
Formatted: Bullets and Numbering
2. LB should be at room temperature while inoculating
3. Spreader should be sterilized to prevent cross contamination while spreading
4. Prepare competent cells only in the Laminar
5. It is essential that the concentrations of the antibiotics added in each plate be uniform.
Hence maintain caution while adding Amp and Tet to LB Broth before pouring plates.
POINTS TO PONDER:
1. Carbenicillin can be used as an alternative to Ampicillin. Why?
2. What are satellite colonies?
REFERENCES:
Working with DNA. Pg 141 to 143
Sambrook and Russel
http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/maps/pBR322_map.pdf
Formatted: Bullets and Numbering
