Biomedical Engineering Reference
In-Depth Information
ing up the hME Reaction Products” in the
MassARRAY Liquid Handler User's
Guide
(Sequenom) (all Sequenom manuals mentioned in this chapter come with
the Sequenom MALDI-TOF MS system).
1.
Place sufficient SpectroCLEAN resin onto a 96-well SpectroCLEAN plate, and
then use a scraper to spread the resin into the wells of the SpectroCLEAN plate.
Next, scrape off the excess resin.
2.
Place a clean, 96-well microplate upside-down over the SpectroCLEAN plate
and align all the wells. Flip the two plates over so that the resin is transferred
from the SpectroCLEAN plate to the 96-well plate. Tap on the SpectroCLEAN
plate, if necessary, to fully release the resin.
3.
Add 70
µ
L water to each well of the 96-plate with resin and thoroughly mix with
the resin.
4.
Add 16
L resin/water solution to each well of the 384-plate from the primer
extension reaction. Seal the plate and place on a rotator for at least 5 min at room
temperature to ensure that the resin and the solution are mixed thoroughly.
µ
5.
Centrifuge the plate for 3 min at 600
g
.
3.2.5. SpectroCHIP Spotting
This step transfers approx 10 nL of the final reaction solution after cation
removal onto a 384-format SpectroCHIP. For the most accurate results, it is
recommended that a 384-plate be spotted onto 2-4 384-format SpectroCHIPs.
Please refer to “Dispensing MassEXTEND Reaction Products onto Spectro-
CHIPs” in the
MassArray Nanodispenser User's Guide
(Sequenom) for
instructions.
3.2.6. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass
Spectrometry
The Quantitative Gene Analysis software is bundled with the MALDI-TOF
MS as a complete package. The software performs data acquisitions, mass spec-
trometric peak identifications, noise normalizations, and peak area analyses.
Please refer to the “SpectroACQUIRE” chapter in the
MassARRAY Typer
User's Guide
for MALDI-TOF MS operations.
4. Notes
1.
The AssayDesigner software (Sequenom) is tailored for designing such assays.
In practice, a user only needs to create an artificial mutation (C/G or G/C) in the
middle of the DNA sequence of interest and input this information into the soft-
ware. The AssayDesigner can also handle multiplex assays, ensuring that the
molecular weight values of all primers (and the extension products derived from
them) involved do not interfere with each other in a mass spectrum.
2.
The quality of extension primers is crucial for accurate quantifications. Specifi-
cally, the (n-1), (n-2), and other by-products resulting from incomplete oligo-
