9 In Situ Hybridization for RNA: Nonradioactive Probe: ds cDNA Probe
133
4. Boil the solution for 10 mins to terminate the reaction.
5. Store the digested probe at -20°C before use.
• The restriction endonuclease is incubated in a water bath at Notes
37°C for 30 mins before this procedure. Because the enzyme
tends to change in the activity, it is stored at -20° C in small
aliquots, which is used once and discarded.
• The effect of restriction endonuclease digestion varies with
the length of ds eDNA, so the time of reaction should be optimized by agarose gel electrophoresis of the digested probe.
(the recommended size of probe is less than 200-400 bp).
• The volume of restriction endonuclease added should be less
than 1/10 volume of the final reaction mixture, because proteins in the enzyme may interfere with the hybridization reaction.
• The digestion was also terminated by the addition of 50 mM
EDTA.
• As for the control, lambda phage DNA (DNase I fragments) or
pUC 18 plasmid (DNase I fragments) were used.
• To digest ds eDNA with DNase 1,10 X restriction buffer contains 1 X SSC and SOmM MgCb, and lambda phage DNA is
digested for 10 mins at 37°C.
T -T dimerization
1. Switch on UV lamps at least 15 mins before use.
2. Apply 30-60 III of DNA solution in several drops onto the dish,
and seal it with a quartz cover.
3. Irradiate it for 40 mins.
• This volume is due to the capacity of a quartz dish.
Notes
• The appropriate dose of irradiation is 12000 J/m2 12000 J/m2 =
irradiation for 40 mins with UV lamp (5 J/m2/sec)
• Immediately after use of dishes, rinse them with DDW 3
times. After rinsing, immerse them into absolute ethanol
and dry up.
133
4. Boil the solution for 10 mins to terminate the reaction.
5. Store the digested probe at -20°C before use.
• The restriction endonuclease is incubated in a water bath at Notes
37°C for 30 mins before this procedure. Because the enzyme
tends to change in the activity, it is stored at -20° C in small
aliquots, which is used once and discarded.
• The effect of restriction endonuclease digestion varies with
the length of ds eDNA, so the time of reaction should be optimized by agarose gel electrophoresis of the digested probe.
(the recommended size of probe is less than 200-400 bp).
• The volume of restriction endonuclease added should be less
than 1/10 volume of the final reaction mixture, because proteins in the enzyme may interfere with the hybridization reaction.
• The digestion was also terminated by the addition of 50 mM
EDTA.
• As for the control, lambda phage DNA (DNase I fragments) or
pUC 18 plasmid (DNase I fragments) were used.
• To digest ds eDNA with DNase 1,10 X restriction buffer contains 1 X SSC and SOmM MgCb, and lambda phage DNA is
digested for 10 mins at 37°C.
T -T dimerization
1. Switch on UV lamps at least 15 mins before use.
2. Apply 30-60 III of DNA solution in several drops onto the dish,
and seal it with a quartz cover.
3. Irradiate it for 40 mins.
• This volume is due to the capacity of a quartz dish.
Notes
• The appropriate dose of irradiation is 12000 J/m2 12000 J/m2 =
irradiation for 40 mins with UV lamp (5 J/m2/sec)
• Immediately after use of dishes, rinse them with DDW 3
times. After rinsing, immerse them into absolute ethanol
and dry up.