Automatic DNA Sequencer
DNA sequencers are basically elaborate gel electrophoresis machines mated
to fluorescent marker detectors. Earlier models used flat traditional
gels with the sample replicated into four different lanes. That way, each
lane could be dedicated to detecting one specific fluorescent marker (corresponding
to one nucleotide species). More modern machines can read all four different
markers in the same lane. The model we use in the lab is even more advanced,
using a capillary tube instead of a flat gel to run the electrophoresis;
thus, the quantity of required sample is smaller.
Readily available in kits, this is a generalized buffered solution of
proteolytic enzymes designed to eat the bacterial cell wall and release
the cellular content into the solution. Other proteins are also digested.
small piece of equipment essentially acts as a sophisticated microfilter.
Microconcentrator columns separate macromolecules based on size. The model
used in this lab retains large DNA molecules while smaller molecules such
as nucleotides, primers, and DNA polymerases pass through. The trapped
DNA molecules can be released by centrifuging the column upside down.
PCR machines have become sophisticated, easy to use, and widely available.
It can be argued that this is one piece of equipment that has revolutionized
the study of genetics in recent years. Standard predescribed settings
can give perfectly good results (as we do in this lab), but the researchers
can always customize the cycling program (i.e., number, temperature, duration,
etc.) in case of difficulties.
PCR Master Mix
The "brew" that contains everything necessary to carry out the polymerase
chain reaction to amplify the 16S rRNA gene. Consists of water; a buffer
to keep the mix at the correct pH for the PCR reaction; large quantities
of the four nucleotides adenine, cytosine, guanine, and thymine; large
quantities of oligonucleotide DNA primers that bind the 16S rDNA region
to initiate the replication process; and a heat-stable DNA polymerase
that extends the copy DNA strand.
are conveniently joined and color-coded microcentrifuge tubes. Each tube
is designed to contain a different primer, and the different colors correspond
to forward or reverse replication direction. (See part 4 for details.)
that has become the workhorse of modern biology labs.