Biology Reference
In-Depth Information
of all the samples that have undergone DNA preparation and are ready
for ligation.
In these early steps, much attention is paid to space and motion.
The time and effort required to move samples through the lab is care-
fully monitored and analyzed. The locations of equipment (centrifuges,
freezers, etc.) and lab benches are optimized so that workers can move
the samples quickly and effi ciently through the lab spaces. These pro-
cesses are constantly scrutinized in order to maintain the highest levels
of quality and effi ciency. Incubation times, solution concentrations, and
other details are monitored and modulated to increase yield.
Once the agar plates are incubated, the responsibility of the MBPG
ends and the Core Sequencing Group takes over. Its work can be divided
into six steps. First, in the “picking” step, a large robot designed by a
company called Genetix uses a high-resolution camera to scan the agar
plates to produce digital images. These images are processed by soft-
ware that identifi es colonies that are good candidates for picking—that
is, colonies that show good growth and are not too close to one another.
A robot arm then moves a 96-pin head over the plate, picking samples
from the selected colonies and transferring each to an individual well of
a 384-well tray or plate containing glycerol solution.
3
The agar plates
are then discarded, and the 384-well plates are incubated for a further
15-18 hours. The samples are then once again placed in a freezer. Sec-
ond, the “TempliPhi” step generates further amplifi cation, using a PCR-
like process to increase the amount of DNA about 10 millionfold in
4 hours. Third, in the “sequencing” step, specially designed nucleotides
tagged with fl uorescent dyes are added to the samples and are incorpo-
rated into the DNA molecules. These incorporations are the sequencing
reactions themselves, the results of which are detected in step 6. In order
to get a sense of what is involved in this process, I quote from a detailed
description:
The sequencing team starts by transferring the DNA from the
TempliPhi Eppendorf Twintec
TM
plate into two other Eppendorf
Twintec
TM
plates and diluting the DNA mixture with water. The
top of the red plate (the TempliPhi plate) is transferred into the
top and bottom halves of one blue plate, so there are two cop-
ies of DNA in one blue plate. The bottom half of the red plate
is transferred into another blue plate, and the red plate is then
discarded. Once this is done, two types of dye are added to each
blue plate; “forward” sequencing dye is added to the top half of
