Biomedical Engineering Reference
In-Depth Information
8.
L of sterile H 2 O to each tube to dilute the labeling reaction to a final
volume of 37.5
Add 33.75
µ
µ
L.
9.
Store at -20°C freezer. The probes are good for 30 d.
3.7. Hybridization (4,10)
1.
Use standard precautions against radioactive materials, solutions, and waste.
2.
Remove the prehybridization tubes from the hybridization oven and place them in
the order as listed next in the Membrane column.
3.
In a separate rack, thaw 10
M cold ASO, and place them in the order as to be
added (cold probe column in Table 5 ) to each pre-hybridization tube; for example,
the A3243G mutant cold ASO is to be added to the tube and membrane labeled
3243nl ( see Note 4 ).
µ
4.
Check the order of the hybridization tubes (same as the prehybridization tubes)
and the ASO tubes, make sure they are in correct order and position before the
addition of hot labeled and cold ASO.
5.
Add 20
M ) to each tube. For example, if
the normal ASO is used as hot probe, the mutant ASO should be used as cold to
reduce nonspecific hybridization ( see Note 4 ).
µ
L of appropriate cold ASO (stock 10
µ
6.
Put the 10
µ
M cold ASO in freezer.
7.
Thaw the hot labeled ASO probes. Place them in the same order as the order of the
name in the membrane and the hybridization tube to be added (hot ASO probe
column in Table 5 ).
8.
Add 10
L of radioactive-labeled hot ASO to each corresponding tube, making
sure that the correct ASO is added to the correct hybridization tube. When adding
the hot probe, add the probe to the hybridization solution and shake the tube gen-
tly to mix well. Do not drop the hot probe onto the membrane directly. It will
cause high background as a result of local high concentration.
µ
9.
Hybridize blots at 65°C for 30 min then turn temperature down to 34°C. Let
hybridize until the temperature reaches 34°C and continue at 34°C for at least 3 h.
3.8. X-Ray Autoradiography
1.
Decant the hybridization buffer to radioactive liquid waste container.
2.
Rinse filters twice in 5X SSC at room temperature ( see Note 5 ).
3.
Wash filters twice in 2X SSC for 30 min at 34°C.
4.
Use a Geiger counter to check for background on the filters. Wash the filters that
have high background in 2X SSC for additional 30 min at 38°C.
5.
Recheck for background and, if necessary, continue to wash at higher temperature
by 2-4°C increment.
6.
Expose filters to X-ray film.
7.
Label the film according to the order of the dot blot membranes.
3.9. Results and Data Analysis
Normal mtDNA hybridizes to the normal probes only. Samples with
homoplasmic mutations hybridize with mutant probes only. Heteroplasmic
mutations hybridize with both normal and mutant probes. Figure 1 depicts an
example of results.
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