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Cloning Class
Definitions:
Clone: a plant or animal that is grown from one
cell of its parent and that has exactly the same
genes as its parent.
Molecular cloning: a set of experimental
methods in molecular biology that are used to
assemble recombinant DNA molecules and to
direct their replication within host organisms.
Transformation: the genetic alteration of a cell
resulting from the direct uptake and
incorporation of exogenous genetic material
(exogenous DNA) from its surroundings and
taken up through the cell membrane(s).
Plasmid: a small circle of DNA, typically found
in bacteria, that is separate from the majority of
bacterial DNA located in the nucleoid.
Selection Marker: a novel trait conferred on
the bacteria that allows you to select for
successful transformants. Eg. Colorimetric or
antibiotic resistance.
Transforming Competent Cells Protocol (E. coli DH5α cells from Life Technologies)
https://tools.thermofisher.com/content/sfs/manuals/subcloningefficiencydh5alpha_man.pdf
1. Thaw on ice one tube of DH5α™ cells. Place 1.5 ml microcentrifuge tubes on wet ice.
2. Gently mix cells with the pipette tip and aliquot 50 µl of cells for each
transformation into a 1.5 ml microcentrifuge tube.
3. Refreeze any unused cells in the dry ice/ethanol bath for 5 minutes before returning
to the -80°C freezer. Do not use liquid nitrogen.
4. Add 1 to 5 µl (1-10 ng) of DNA to the cells and mix gently. Do not mix by pipetting up
and down. For the pUC19 control, add 2.5 µl (250 pg) of DNA to the cells and mix
gently.
5. Incubate tubes on ice for 30 minutes.
6. Heat shock cells for 20 seconds in a 42°C water bath without shaking.
7. Place tubes on ice for 2 minutes.
8. Add 950 µl of pre-warmed medium of choice to each tube.
9. Incubate tubes at 37°C for 1 hour at 225 rpm.
10. Spread 20 µl to 200 µl from each transformation on pre-warmed selective plates.
We recommend plating two different volumes to ensure that at least one plate will
have well-spaced colonies. For the pUC19 control, plate 100 µl on an LB plate
containing 100 µg/ml ampicillin.
11. Store the remaining transformation reaction at +4°C. Additional cells may be plated
out the next day, if desired.
12. Incubate plates overnight at 37°C.
What is cloning used for?
We need cloning in order to conduct
basic genetic manipulation. This
particular technique, transformation, is
used to get the DNA inside the bacteria
and to produce multiple copies. Once
the DNA is inside the bacteria, the
bacteria will continuously amplify the
DNA, given the correct sequences. On
the one hand, this process allows us to
cement our recombinant DNA, and on
the other hand this process allows us to
amplify the DNA so we have enough for downstream applications.
So how does transformation work?
Again, the whole process of transformation is
getting the DNA inside the bacteria. The
bacteria won’t readily uptake the DNA, so we
have to prepare the bacteria first to coax it
into taking up the DNA. There are two
methods of coaxing the cell: The first is
chemically competent transformation and the
second is electroporation. The two methods
are similar in their outcome but slightly
different in their approach. The idea is that
we need to make holes, or pores, in the membrane of the cell so the DNA has a greater
chance to enter the cells.
We are working with chemically competent
cells. In order for the DNA to have a better
chance of floating into the cell, we need to
bring the DNA close to the cell membrane.
However, the cell membrane is negatively
charged and the DNA is negatively charged,
so they naturally repel each other! In order to
overcome this challenge, we incubate the
cells with a Calcium Chloride (CaCl) salt. The positively charged Calcium ions will be
attracted to the membrane and the DNA will be attracted to the Calcium ions, so we end up
with a Membrane-Calcium-DNA sandwich! That way, when we heat shock the cells to create
pores, the DNA will be very close and is much more likely to float in.
Why do we incubate it at 37 degrees Celcius for an hour before plating them?
The plate has an antibiotic on it that will kill everything without resistance to that specific
antibiotic. The idea is that the bacteria cells do not have an innate resistance to the
antibiotic so they would be killed upon contact. The plasmid DNA will usually contain a
gene that will confer antibiotic resistance upon the cell, allowing the bacteria to survive in
the presence of the specific antibiotic. However, once you transform the bacteria, the cell
requires time to express the antibiotic resistance gene and build up resistance. If you were
to plate the bacteria immediately, they would all die because they have not built up the
resistance. That’s why you must grow the newly transformed cells first for an hour before
plating them.
What are the next steps?
If you want to do more sophisticated genetic manipulation, you would isolate a colony from
your plate (to get a pure genetic line), grow it up, isolate the plasmid DNA, and use it for
further applications. The idea is that you can take multiple plasmids, digest (cut) them in
specific ways, and ligate (paste) them together to develop new functions. For example, if
you were to ligate together a gene that glowed green with a gene that detects arsenic and
transform them into bacteria, you will have created a biosensor that glows green whenever
the cell detects arsenic.