Biomedical Engineering Reference
In-Depth Information
24 Vortex twice for 5 s each.
25 Centrifuge at 13 000
g
for 10 s to collect all the liquid in the bottom of the tube.
26 Place a NucleoSpin
®
Tissue XS column into a 2 ml collecting tube, and apply the sample
to the column.
27 Centrifuge at 13 000g for 1 min.
28 Discard the flow-through.
29 Place the column into a new 2 ml collecting tube.
30 Add 50
μ
l of buffer B5 to the membrane.
31 Centrifuge for 1 minute at 11 000
g
.
h
32 Add 50
μ
l of buffer B5 directly onto the membrane.
33 Centrifuge at 13 000g for 2 min.
34 Place the column into a 1.5 ml microcentrifuge tube.
35 Apply 50
μ
l of buffer TE directly onto the center of the silica membrane of the
column.
i
Centrifuge at 13 000
g
for 1 min.
36
Notes
g
This protocol can be used instead of the non-column based method described in Protocol 3.1.
Protocol 3.2 is more expensive than Protocol 3.1 and the yield of DNA is slightly lower. However,
the purity of the DNA isolated is higher.
h
It is not necessary to discard the flow-through. Reuse the collecting tube.
i
For a higher DNA yield, repeat step 35 by pipetting the eluate from step 36. back onto the
column.
PROTOCOL 3.3 DNA Concentration Measurement
Using Picogreen
Equipment and reagents
•
TE: 10 m
M
Tris-HCl, pH 8.0, 0.1 m
M
EDTA
Picogreen reagent (Molecular Probes)
j
•
•
Lambda DNA standards
Microtiter plates (Dynex Immulux
TM
)
•
Fluorescence plate reader
•
Centrifuge for microtiter plates
•
