Biomedical Engineering Reference
In-Depth Information
2. Materials
2.1. RNA Extraction
1.
QIAamp viral RNA Mini Kit (Qiagen, Hilden, Germany).
2.
Absolute ethanol.
2.2. Reverse-Transcription
1. Superscript III RNase H
-
reverse transcriptase (Invitrogen, Carlsbad, CA).
2. Random hexamers (Applied Biosystems, Foster City, CA).
3. 5X First-strand synthesis buffer: 250 m
M
Tris-HCl, pH 8.3, 375 m
M
KCl, 15 m
M
MgCl
2
(Invitrogen).
4. RNasin RNase inhibitor (Promega, Madison, WI).
5.
dNTP (Invitrogen).
6.
0.1
M
Dithiothreitol (DTT).
7.
RNase-free water (Promega).
2.3. PCR Amplification
1.
Advantage cDNA Polymerase mix and buffer (BD Biosciences Clontech,
Palo Alto, CA).
2.
dNTP.
3.
PCR primers.
4.
Distilled water.
2.4. Genomic Sequencing
1.
BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems).
2.
ABI Prism 3100 Genetic Analyzer (Applied Biosystems).
3.
DYEnamic ET Dye Terminator Kit (GE Healtcare-Biosciences, Little Chalfont, UK).
4.
MegaBACE 1000 Sequencing System (GE Healthcare-Biosciences).
2.5. Sequence Analysis and Comparison
SeqScape software (Applied Biosystems).
3. Methods
3.1. Precautions Against Potential Contamination
Genomic sequencing involves PCR amplification, which produces numer-
ous copies of the target DNA, and cycle sequencing, which requires the
pipetting and manipulation of PCR products. These steps could easily con-
taminate the laboratory environment with amplified products. Such contami-
nation problems would affect the interpretation of sequencing results, and
adversely affect the performance of diagnostic tests designed to detect the same
viral sequences. Hence, extreme care should be taken to avoid contamination.
We suggest the following precautions:
