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Analyzing Data
Transformation
• The DNA molecule is hydrophilic (water-soluble) but cell
membranes are made of a very hydrophobic lipid bilayer.
Two means of artificial transformation commonly used in
labs: electroporation and chemical transformation.
• During electroporation, short bursts of current are
passed through a solution containing bacteria at high
voltage. The current makes the cell membrane leaky
(porous) for a short time, allowing the cells to take up
DNA molecules from the solution.
• In chemical transformation, bacteria are exposed to
solutions which alter their cell membranes enough to
make the DNA molecules pass through and into the cell.
Chemical transformation procedures sometimes also use
a heat shock treatment.
Transformation is Inefficient!
• Most bacteria in the solution WILL NOT
become transformed.
• Identify the needles in the haystack of
bacteria.
– Antibiotic resistance genes on antibiotic plates
– Other forms of selection in yeast like genes
for amino acid synthesis.
Chemical Transformation
• Escherichia coli cells are grown to log phase. Cells are
concentrated by centrifugation and resuspended in a
solution containing CaCl2
• Exposure to calcium ions renders the cells able to take
up DNA, or competent.
• Plasmid DNA is mixed with the cells and presumably
adheres to them.
• The mixture of DNA and cells is then heat shocked,
which allows the DNA to efficiently enter the cells.
• The cells are grown in nonselective medium to allow
synthesis of plasmid-encoded antibiotic resistance
proteins, then plated on antibiotic-containing medium to
allow identification of plasmid-containing colonies.
Important Considerations
• Competent are relatively easy to make fresh and
harder to freeze- need to be quick frozen to
preserve viability.
• Competent cells need to be kept in -80° C
freezer and kept on ice. They are good for
approximately 6 months at the highest efficiency
and then gradually lose viability.
• Competent cells should be used immediately
after thawing. Remaining cells should be
discarded rather than refrozen.
Factors That Affect
Transformation Efficiency
• DNA Concentration
– More DNA is not better- Transformation increases
linearly for a while and then plateaus at about 10 ng.
We used 0.1 ng DNA in 1 ul.
– When comparing competent cells, use the same DNA
for all transformations
• Forms of DNA
Linear and single-stranded DNA transforms <1%
as efficiently as supercoiled DNA.
– Plasmid size- bigger isn’t better.
– We used supercoiled DNA and most companies do
too to calculate transformation efficiency.
Factors That Affect
Transformation Efficiency
• Purity of DNA
– For electroporation, never use more than 1 µl plasmid DNA per
transformation. The salts contributed by the preparation can
cause low transformation efficiencies. The sample DNA to be
transformed by electroporation must be in a low-ionic-strength
buffer, such as TE buffer or water. DNA samples containing too
much salt will cause arcing at high voltage, possibly damaging
both the sample and the machine.
– Column-purified DNA is generally free of contaminants that
would interfere with chemical transformation.
– Contaminants in miniprep DNA can interfere with
transformation. Limit the amount of miniprep DNA in a
transformation as much as possible and never use more than 5
µl per 50-µl reaction.
Factors That Affect
Transformation Efficiency
• Ligation mixtures inhibit transformation.
– Ironic! That is why people start with very competent
cells.
– Ligase strongly inhibits electroporation, but this effect
can be limited by heat inactivating the ligase in the
ligation mixture (65°C for 5 min) prior to adding DNA
to the cells. Dilute the ligation mixture to dilute the
salts.
Factors That Affect
Transformation Efficiency
• Heat Shock
– The heat shock step can affect transformation
efficiency. Optimal efficiencies are obtained
with a thin-wall tube such as a PCR tube or
another thin-walled tube (42°C for 45 sec).
With thick-walled tubes, such as a
microcentrifuge tube, the optimal parameters
are slightly different (37°C for 60 sec). When
in doubt, it is better to use the 60 second
shock at 37°C.
Factors That Affect
Transformation Efficiency
• Length of Time After Transformation
– The effect of the expression time depends on the
plasmid and the strain.
– Longer expression times give higher numbers of
colonies, but since they are siblings, the number of
transformants is not higher.
Factors That Affect
Transformation Efficiency
• SOC Medium
– SOC Medium is highly recommended as the expression
medium. SOC Medium gives two-fold better results than LB
Medium for chemically competent cells.
• Selective Plates
– Some batches of agar plates are better than others.
– Fresh plates are best!
– Plating large numbers of cells on selective plates or for extended
periods of incubation may allow growth of satellite colonies
which are breakthrough colonies because of poor antibiotic
stability.
– The colonies can be identified by streaking them on the selective
agar plates.
– If they are true transformants, they will grow.
– If they are satellites, they will not grow.
Why this Matters
• Transformation efficiency is a measure of the
amount of cells within the bacterial culture that
are able to take up DNA molecules.
• For some molecular biology projects, such as
cloning and subcloning, high transformation
efficiency is not critical.
• However applications such as construction of
genomic libraries require that the bacteria have
very high transformation efficiency.
Controls
• We are going to calculate transformation
efficiency by counting bacteria on our
plates
• Count the CPlasmid plates # of colonies.
• Count blue and white separately
• Average the 1/10 dilutions colony
numbers.
Blue-White Cloning
• The is a molecular technique that allows for the
detection of successful ligations in vector-based
gene cloning. Works in bacteria plasmids, phage
cloning and yeast.
• DNA is ligated into a vector. The vector is then
transformed into competent cells. The
competent cells are grown in the presence of Xgal. If the ligation was successful, the bacterial
colony will be white; if not, the colony will be
blue. This technique allows for the quick and
easy detection of successful ligation, without the
need to individually test each colony.
Blue-White Cloning
• X-gal is a colorless modified galactose sugar that is
metabolized by β-galactosidase to form an insoluble
product (5-bromo-4 chloroindole) which is bright blue.
• Isopropyl β-D-1-thiogalactopyranoside (IPTG), functions
as inducer of the Lac operon, and is used to enhance
the blue color, although it isno always needed.
• The hydrolysis of colorless X-gal by the β-galactosidase
causes the characteristic blue color in the colonies; it
shows that the colonies contain vector without insert.
• White colonies indicate insertion of foreign DNA and loss
of the cells' ability to hydrolyze the marker.
BW Cloning requires the correct
bacterial strain is used.
Calculating Cloning Efficiency
• http://www.sciencegateway.org/tools/transf
orm.htm
• Linear DNA fragments electrophoreses through the
agarose gel at a rate that is inversely proportional to the
log10 of their molecular weight.
• For convenience, molecular weight is expressed as
numbers of base pairs (bps).
• Use a ruler on your gel or a photograph of your gel to
measure the distance each band of DNA has migrated
from the sample well, and construct a graph that relates
distance migrated to base-pair size of the fragment.
• Graph a “standard” ( fragments of known molecular
weight) on semilog graph paper, and then use the plot to
derive the bp size of fragments of unknown size that
have run on the same gel side by side with the standard.
Hind III
HindIII bp
size
(standard)
23,130 bps
Measure the known sizes
first in MM migration.
Plot these on a semilog
excel spread sheet.
Measure the unknown
bands MM in migration.
9416
6557
4361
2322
2027
564
Estimate their sizes based
on the plot you made.