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Transformationandtransfectionfinal 180609201420 PDF
Transformationandtransfectionfinal 180609201420 PDF
CaCl2
CaCl2
Transformation - Mechanisms
• Bacteria - Artificial competence - Temperature
– Chilling cells in the presence of divalent cations such as Ca2+
(in CaCl2) prepares the cell walls to become permeable to
plasmid DNA.
– Cells are incubated with the DNA and then briefly heat
shocked (42oC for 30-120 seconds), which causes the DNA to
enter the cell.
– This method works well for circular plasmid DNAs but not
for linear molecules such as fragments of chromosomal
DNA.
– An excellent preparation of competent cells will give ~10 8
colonies per μg of plasmid. A poor preparation will be about
104/μg or less. Good non-commercial preps should give 105 to
106 transformants per microgram of plasmid.
The CaCl2 method
This method also alters the permeability of the cell membrane:
• Ca2+ interacts with the negatively charged phospholipid heads of the
cell membrane, creating an electrostatically neutral situation.
• Lowering the temperature stabilizes the membrane, making the
negatively charged phosphates easier to shield.
• Then a heat shock creates a temperature imbalance and thus a
current, which helps get the DNA into the cell.
Transformation - Mechanisms
A plasmid again?
• A plasmid DNA molecule contains sequences allowing it to be
replicated in the cell independently of the chromosome.
• Plasmids used in experiments will usually also contain an
antibiotic resistance gene which is placed in a bacterial strain
that has no antibiotic resistance.
• Therefore, only transformed bacteria will grow on a media
containing the antibiotic.
Transformation - Mechanisms
• Bacteria - Artificial competence –
Electroporation
– Electroporation is another way to make holes in cells, by
briefly shocking them with an electric field of 100-200 V/cm.
– Now plasmid DNA can enter the cell through these holes.
– Natural membrane-repair mechanisms will close these holes
afterwards.
Electroporation!
The general idea behind electroporation is that by applying a
short electrical pulse to the cells, we can alter membrane
conductivity and permeability. It is more effective than the CaCl 2
method (chemical competence).
normal
DNA is a negatively
Polar molecules charged molecule due
don’t normally to phosphate groups (in
cross the cell its “backbone”).
membrane easily
because the
inside is
hydrophobic.
But
electroporation
makes pores in
the membrane
that are electroporated –
hydrophilic, hydrophilic pore
enabling DNA to
pass through.
To make electrocompetent cells:
1. Inoculate a colony into ~50 ml (no salt) LB and grow at 37°C
overnight.
2. Add ~25 ml culture medium into 1 L LB medium.
3. Grow the cells at 37°C in a shaking incubator.
4. Grow cells to an A600 of ~0.6-0.7. This represents the bacteria’s
log-phase growth. Why log phase? Cells in this phase are
growing rapidly, and are healthy and uniform. (Also keep in
mind that since they divide so rapidly, you should work at a
decent pace.)
5. Pour ~250 mL into a tube and spin down in a centrifuge at 4°C.
6. Remove supernatant and resuspend cells in dH2O.
7.Repeat centrifugation / removal of
supernatant several times.
8.Resuspend in 10% glycerol and keep
in freezer until ready to use.
Dendrimers
•Dendrimers are highly
branched molecules that form a
complex with DNA
CELL PREPERATION:
1. Plate cells on a glass coverslip.
2. For a good injection, a 60-80% cell confluence at the day of injection is
required.
3. The day of the experiment transfer each coverslip in a 6 cm diameter
plate with 5 ml of medium/plate
DNA PREP
1. Dilute the DNA in ddH2O to a final concentration of 20-150 ng/µl
2. Centrifuge 15 min. at 13,000 rpm RT and transfer the supernatant in a
new clean eppendorf tube.
3. You can mix different DNA but the final concentration has to be 150
ng/µl total max. Alternatively IgGs can be mixed to the DNA in order
to use them as microinjection efficiency marker.
4. Inject the sample into target cell nuclei
A Quick Note:
Generally, it is good practice to do a control transformation
(with water) just to aid any future necessary troubleshooting. If
you get colonies on the control plate, something definitely
went wrong with your transformation.
?
Questions???
Thanks