9 In Situ Hybridization for RNA: Nonradioactive Probe: ds cDNA Probe
129
with a restriction endonuclease. This chapter describes the protocol for preparation of a T -T dimerized ds cDN A probe and detection of mRNA transcripts in tissue sections.
Materials
Restriction endonuclease cleavage is accomplished simply by incubating the enzyme with ds cDNA in appropriate reaction conditions. The amounts of enzyme and DNA, the buffer ionic concentrations, and the temperature and duration of the reaction
will vary depending upon the specific application.
- ds cDNA probe
- Restriction endonuclease
- lOx restriction endonuclease buffer
- TE buffer (pH 7.4); 10mM Tris-HCl (pH 7.4), 1mM EDTA
- 50 mM EDT A (optional)
When an appropriate enzyme cannot be found out, we use
DNase I with the following buffers.
- ds cDNA probe
DNase I (Sigma)
Ix SSC (standard saline citrate); 0.15 M NaCI, 0.015 M sodium
citrate (pH 7.0)
50 mM MgCl2
TE buffer (pH 7.4); lOmM Tris-HCI (pH 7.4), 1mM EDTA
50 mM EDT A (optional)
UV lamp (254 nm wave length) (Toshiba Ltd., Tokyo, Japan)
UV radiometer (Tokyo, kougaku Kikai, Ltd., Tokyo, Japan). It
is required for taking an accurate measurement of 5 J/m2/sec
Quartz dish (GL Sciences, Ltd., Tokyo, Japan)
Forceps for handling a quartz dish
- Paraformaldehyde (PFA) (MERCK)
- Proteinase K (Wako); Dissolve the proteinase K at 1 mg/ml
concentration in DDW. Store at - 20°C in small aliquots.
- Mixed bed resin (Bio-Rad)
- Formamide (nacalai tesque) (for prehybridization); Formamide used for prehybridization must be of the highest grade
commercially available (nacalai tesque) and be deionized
with mixed bed resin just before use. (Mix 50 ml of formam ide
and 5 g of mixed bed resin and stir for 30 mins.)
for digestion
of ds cDNA
probe with
restriction endonucleases
Notes
for T-T dimerization
for in situ
hybridization
(direct
method}
129
with a restriction endonuclease. This chapter describes the protocol for preparation of a T -T dimerized ds cDN A probe and detection of mRNA transcripts in tissue sections.
Materials
Restriction endonuclease cleavage is accomplished simply by incubating the enzyme with ds cDNA in appropriate reaction conditions. The amounts of enzyme and DNA, the buffer ionic concentrations, and the temperature and duration of the reaction
will vary depending upon the specific application.
- ds cDNA probe
- Restriction endonuclease
- lOx restriction endonuclease buffer
- TE buffer (pH 7.4); 10mM Tris-HCl (pH 7.4), 1mM EDTA
- 50 mM EDT A (optional)
When an appropriate enzyme cannot be found out, we use
DNase I with the following buffers.
- ds cDNA probe
DNase I (Sigma)
Ix SSC (standard saline citrate); 0.15 M NaCI, 0.015 M sodium
citrate (pH 7.0)
50 mM MgCl2
TE buffer (pH 7.4); lOmM Tris-HCI (pH 7.4), 1mM EDTA
50 mM EDT A (optional)
UV lamp (254 nm wave length) (Toshiba Ltd., Tokyo, Japan)
UV radiometer (Tokyo, kougaku Kikai, Ltd., Tokyo, Japan). It
is required for taking an accurate measurement of 5 J/m2/sec
Quartz dish (GL Sciences, Ltd., Tokyo, Japan)
Forceps for handling a quartz dish
- Paraformaldehyde (PFA) (MERCK)
- Proteinase K (Wako); Dissolve the proteinase K at 1 mg/ml
concentration in DDW. Store at - 20°C in small aliquots.
- Mixed bed resin (Bio-Rad)
- Formamide (nacalai tesque) (for prehybridization); Formamide used for prehybridization must be of the highest grade
commercially available (nacalai tesque) and be deionized
with mixed bed resin just before use. (Mix 50 ml of formam ide
and 5 g of mixed bed resin and stir for 30 mins.)
for digestion
of ds cDNA
probe with
restriction endonucleases
Notes
for T-T dimerization
for in situ
hybridization
(direct
method}