Protocols and Applications Guide (US Letter Size) - Promega

||||||||| 9DNA Purification
D. MagneSil® Genomic Systems for Blood Isolation
Promega offers several MagneSil® paramagnetic
silica-based systems for specialized sample-type use. Three
systems address isolation of DNA from whole blood on
automated platforms: MagneSil® ONE, Fixed Yield Blood
Genomic System; MagneSil® Blood Genomic, Max Yield
System and MagneSil® Genomic, Large Volume System.
These DNA purification systems are for automated use and
require hardware accessories in addition to the instrument
workstation. A list of the essential accessories for use with
a robotic setup are included on the online catalog pages for
each DNA isolation system at: www.promega.com. The
main differences in these three DNA purification systems
are format, the blood volume processed and DNA yield
recovered. The MagneSil® ONE, Fixed Yield Blood Genomic
System (Cat.# MD1370) purifies 1µg of DNA (±50%) from
60µl of anticoagulated whole blood in a 96-well plate
format. Purification of a "fixed yield" of DNA eliminates
the need to quantitate and normalize concentrations
post-purification.
To maximize the quantity of DNA recovered from 200µl
blood, use the MagneSil® Blood Genomic, Max Yield
System (Cat.# MD1360). The methodology is the
same—lysing the cells and capturing the genomic DNA
from the solution—but the recovered yield is more variable,
between 4–9µg, depending on the number of white cells.
Figure 9.13 shows a multiplex PCR using DNA isolated
from both the MagneSil® Blood Genomic, Max Yield
System, and the MagneSil® ONE, Fixed Yield Blood
Genomic System.
Multiplex A Multiplex B
M Max Fixed Max Fixed M
Figure 9.13. Multiplex PCR analysis on genomic DNA purified
from blood. Genomic DNA purified with either the MagneSil®
Blood Genomic System, Max Yield System (Max) or the MagneSil®
ONE, Fixed Yield Blood Genomic System (Fixed) was amplified
using the Y Chromosome Deletion Detection System, Version 1.1.
Ten microliters of the amplification reactions for Multiplex A and
B was run on a gel and visualized by ethidium bromide staining.
For the ability to isolate large quantities of genomic DNA
from large-volume tubes including blood samples (1–10ml),
the MagneSil® Genomic, Large Volume System (Cat.#
A4080, A4082, A4085) may meet your needs. There are
several accessories needed in order to use this system for
isolation of genomic DNA (see the Specialized Genomic
DNA Purification Protocol for a listing), but the MagneSil®
Genomic, Large Volume System, can process even
mishandled blood samples and, depending on the white
cell count, may yield ~450µg genomic DNA/10ml blood.
These DNA isolation systems produce high-quality DNA
suitable for use in PCR, multiplex PCR and SNP genotyping
Protocols & Applications Guide
www.promega.com
rev. 3/09
4211TA06_3A
applications. As seen in Figure 9.14, genomic DNA isolated
using the MagneSil® Genomic, Large Volume System,
works well in real-time PCR analysis.
A.
R n
10 1
10 0
10 -1
10 -2
B.
35
C t 30
0
5µl DNA
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Cycle
25
20
15
10
5
0
0.001 0.01 0.1 1 10
Input DNA (µl)
Figure 9.14. Real-time PCR assay for quality of genomic DNA
purified by the MagneSil® Genomic, Large Volume System.
Decreasing volumes of isolated human DNA were analyzed using
β-actin real-time PCR control reagents from Applied Biosystems.
Panel A. Amplification curve for the DNA volume range tested.
Panel B. Linear detection for the amplification.
While these MagneSil® Genomic Systems are primarily
designed for use with whole blood, other sample types can
be used. However, there are no specific protocols associated
with other sample types. Visit our Citations database
(www.promega.com/citations/) or contact Promega
Technical Services (techserv@promega.com) to learn about
other possible sample types used for genomic DNA
purification.
Additional Resources for the MagneSil® Genomic
Systems for Blood Isolation
Technical Bulletins and Manuals
TB313 MagneSil® ONE, Fixed Yield Blood Genomic
System Technical Bulletin
(www.promega.com/tbs/tb313/tb313.html)
TB312 MagneSil® Blood Genomic, Max Yield System
Technical Bulletin
(www.promega.com/tbs/tb312/tb312.html)
TB549 MagneSil® Genomic, Large Volume System
Technical Bulletin
(www.promega.com/tbs/tb549/tb549.html)
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PROTOCOLS & APPLICATIONS GUIDE 9-21