John M. S. Bartlett.pdf - Bio-Nica.info

Cycling Primed In Situ Amplification 429
3.4. Microwave Pretreatment
1. Place about 10 anti-bumping granules in a 50-mL Coplin jar and add the microscope slides.
Slides can be placed back to back so that a jar contains 10 slides.
2. Fill the jar with 10 mM Tris-HCl, 5 mM EDTA (40–50 mL).
3. Place the jar in the center of the microwave oven, switch on full power until boiling,
and boil for a further 50 s.
4. Quickly transfer the slides to a staining jar containing 70% ethanol at room temperature.
Leave for 1 min.
5. Pass through 90% and 100% ethanol for 1 min each. Slides can either be air dried for
immediate use or stored at 4°C in 100% ethanol for several months if desired.
3.5. Cycling PRINS
Reaction volumes will vary with your system (see Note 8). As a rough guide,
22- × 22-mm coverslips require about 15 µL, and 50- × 22-mm coverslips or Amplicovers
require about 40 µL.
1. Prepare 100-µL reaction mix as follows: 2 µL of dNTP mix, 0.4 µL of DIG-dUTP, 10 µL of
10× PCR buffer, 2 µL (10 units) of AmpliTaq Gold DNA polymerase, 2 µL oligonucleotide
primer, and 83.6 µL water.
2. Place 15 to 40 µL on the slide area, according to the cell preparation area and size of your
coverslip, or 40 µL for Amplicovers.
3. For coverslips, seal with rubber solution and allow this to dry (a fan or laminar flow cabinet
speed this up). For Amplicovers, follow manufacturers’ instructions.
4. Transfer the slides to a flat block thermal cycler. A suitable program for the primers
described is: 18 min at 95°C (to activate the AmpliTaq Gold DNA polymerase: reduce
to 5 min for standard Taq), then 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min
for 15 cycles (see Note 8).
5. Peel off rubber glues, or take off Amplicovers, and immerse slides in a Coplin jar containing
stop buffer at 65°C. Coverslips may come off with the glue, if not they will fall off readily
in the stop bath. After 1 min, transfer slides to a jar containing wash buffer (we have also
used 2× SSC, 45°C for 5 min to stop the reaction).
3.6. Detection
Do not allow slides to dry during this process.
1. Prepare blocking buffer, and pipet 40 µL on to a clean cover slip. Shake the slide free
of excess wash buffer, then pick up the cover slip with slide. Leave the slide at room
temperature for 5 min.
2. Dilute the anti-DIG FITC 1/100 in blocking buffer (e.g., add 500 µL of blocking buffer
to a 5-µL aliquot. This is usually a suitable dilution, although batch-to-batch variation
may be encountered).
3. Remove the coverslip from the slide and drain the excess fluid by shaking or briefly
blotting one edge against an absorbent paper towel. Pipet 40 µL of diluted anti-DIG FITC
on to the coverslip and replace the slide. Incubate in a moist chamber (e.g., a sandwich
box lined with damp filter paper) at 37°C for 30 min.
4. Wash the slides 3× for 2 min in prewarmed wash buffer (42°C) in a Coplin jar.
5. Prepare mountant: add 3.75 µL of propidium iodide (see Note 9) stock to 100 µL of
Vectashield.