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Section 2.5.3
Preparation of
32
P-Labeled Probes by
RNA
Transcription
5 X Transcription buffer 4 µl
DTT 100 mM 2 µl
RNAsin (Promega) 1 µl
GTP 10 mM 1 µl
ATP 10 mM 1 µl
CTP 10 mM 1 µl
Linearized Plasmid (1 µg) 2 µl
α 32P-UTP 10 mCi/ml 2 µl
H2O 5 µl
RNA polymerase SP6 or T7, 1 µl
depending on the promoter
contained in the plasmid
Allow to dry completely. Place the dry filter paper inside the lead lid used
for transporting the radioactive nucleotide. Place the Geiger counter on
the lid and measure the radioactivity as shown in the table.
Hybridization
Place the membrane in a suitable plastic bag, seal, and cut a corner. For
a 12 x 16-cm membrane, pour 9.6 ml hybridization buffer into the bag,
reseal the bag, and incubate it in a water bath at 55 oC for 10 minutes.
Mix very well, and seal the plastic bag carefully to avoid bubbles forming.
Incubate overnight in a water bath at 55 oC for viroids and 45 oC for
viruses.
The next day, cut a corner of the plastic bag and remove the hybridization
solution. Be very careful because the solution is highly radioactive.
Dispose of it in an appropriate container.
Washing
All washing steps should be done in a metal tray, using a rotary shaker.
The washing solutions should be discarded after use as indicated in the
warning above.
Washing buffer 1
0.36 M NaCl (21 g/l)
20 mM Tris-base (2.4 g/l)
37% HCl (1.48 ml/l)
20% SDS (5 ml/l)
Washing buffer 2
0.1X SSC
0.1% SDS
Washing buffer 3
2X SSC
The next steps should take place in a dark room with an appropriate red
light (use a Kodak GBX-2, safelight filter), and handlers should wear
gloves.
e) Remove the plate from the cassette in a dark room, and cut one
corner to identify the original position inside the cassette.
Identify the results by placing the photographic films over the membrane
and matching the position of the spots on the films with the positive spots
on the membrane.
Buffer Preparation
a) 20X SSC (l liter)
175.32 g NaCl (3 M)
88.23 g dehydrated sodium citrate (0.3 M).
f) T10E1 + 1% 2-mercaptoethanol
g) 5% TCA
For 100 ml, dissolve 18.6 g EDTA (MW 372.2) in sterile distilled
water. The dissolving process is very slow. Add NaOH in tablets to
adjust pH to 8.0. Add sterile distilled water to adjust final volume to
100 ml.
l) Required solutions