Biomedical Engineering Reference
In-Depth Information
1. Drain size-exclusion chromatography columns by snapping off bottom and
allowing to drain into waste tube.
2. The radioactive target mixture is applied to the column and eluted in 100-
µ
L
fractions using sterile water.
3. The fractions are rapidly assessed directly in the scintillation counter without
scintillation fl uid (“Cerenkov” counting), and provide immediate feedback on
the resolution of target and unincorporated radionucleotide.
4. The three fractions comprising the fi rst radioactive peak serve as the basis for the
hybridization experiment. Successful chromatography will completely separate
this peak from the larger peak of unincorporated radioactivity. Spot 1
L from
each of the three fractions on Whatman fi lter paper in duplicate and determine the
radioactivity by liquid scintillation spectrometery (with scintillation fl uid). This
will provide a much more accurate and high-effi ciency estimate of radioactive
incorporation. Following scintillation counting, the amount of radioactivity per
microliter of fraction can be determined from the average of the replicates.
Optimal hybridization results are achieved using a total of 3.5-8.0 million cpm
per sample. This will generally require pooling two fractions.
µ
5. The two experimental target samples must now be equilibrated so that each
sample has the same number of cpms. Equivalent amounts of radioactivity should
be added to two separate tubes, which are then brought up to an equal volume
and 1
L counted again in duplicate as before. The average sample counts should
be within 1-2% of each other for use in hybridization.
µ
6. Once the radioactivity has been equilibrated, the samples can be stored in a
protective container at -20°C for 1-2 d until the remainder of the experiment is
prepared (i.e., prehybridization of the fi lters).
3.5. Prehybridization
Prior to the actual hybridization experiment, the membranes are prehybrid-
ized to block nonspecifi c interaction with radiolabeled target. The importance
of effective prehybridization is illustrated in
Fig. 3
.
1. Remove array membranes from freezer. Wet membranes in DEPC-treated water
and insert into bottles. The bottles should be well washed and the interior surfaces
coated with a siliconization agent (Sigmacote) to prevent the membranes from
sticking to the glass surface. The membranes should be touched as little as
possible with gloves, using tweezers to hold the edges and manipulate them.
Arrange the membranes in separate bottles with the face of the membrane
containing the probe facing the inside of the tube.
2. Pour off water and briefl y allow to drain.
3. Freshly make prehybridization buffer: 25 mL of the hybridization buffer provided
by Clontech and 250
L of a 10 mg/mL denatured (boiled) salmon sperm DNA
(ssDNA) solution. The buffer must be mixed thoroughly while heating in a
68°C water bath.
µ
Search WWH ::
Custom Search