Biology Reference
In-Depth Information
with commercially available kits. As noted in
Nature Methods
(2011)
, “Modern
molecular biology would be impossible without commercial kits.” These kits are
essential for isolating DNA and plasmids, extracting RNA, conducting restriction
digests, cloning, and many other tasks. Kits save time and may be cost-effective.
Only a relatively few years ago, molecular biologists had to produce their own
enzymes, make buffers, and spend a great deal of time doing very basic bio-
chemistry. However, although kits save valuable time, “The use of kits is blamed
for undercutting researchers' ability to recognize artifacts and for making young
scientists more inclined to trust their results than to question them,” and “Any
self-respecting scientist should be able to say what is happening at every step of
a protocol” (
Nature Methods
2011
).
When using kits, carefully read the methods provided by the manufacturer.
Manufacturers should explain what to do at each step, what is happening, and
what the reagents are. When choosing a kit, consider the quality of the infor-
mation provided and any troubleshooting information. Examine each step
before you begin to be sure you understand what you will be doing, and why.
If you have questions, most companies provide a “hot line” that you can tele-
phone or an email address for advice and information. Because improvements in
kits are made regularly, it is a good idea to contact the manufacturer's website
to check on methods updates; some won't be in the printed directions. Once in
a very rare while, kits will not perform properly because they are defective, so
don't hesitate to contact the company if you have problems.
5.2.2 A Simple Cloning Experiment
The project described here is simple and basic (
Figure 5.1
). It involves inserting a
piece of
exogenous
(or foreign) DNA extracted from one organism into a plas-
mid that has been engineered to serve as a
vector
to carry the exogenous DNA
into
E. coli
. The
E. coli
cells with the exogenous DNA (in the plasmid vector) will
be able to yield large numbers of the desired DNA molecules. Once the DNA has
been multiplied (
cloned
), the exogenous DNA can be studied in detail.
Although the experiment in
Figure 5.1
conceptually is simple, cloning a frag-
ment of foreign DNA into a vector demands that several steps be achieved:
1) The circular vector DNA must be purified and cut. 2) The exogenous DNA
must be extracted, purified, and cut. 3) The vector DNA and exogenous DNA
must be joined together. 4) The reactions should be monitored. 5) The recom-
binant plasmid or vector containing the exogenous DNA must be put back into
E. coli
to be multiplied. 6) The recombinant plasmid then must be removed from
E. coli
and purified for analysis or use of the exogenous DNA.
