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YOSHIO KANEMITSU AND TAKEHIKO KOJI
- SI endonuclease (e.g., Takara, Japan, store at -20°C)
- lOx Sl endonuclease buffer (supplied with enzyme, e.g., containing 300 mM NaCH3COOH pH 4.6, 2.8 mM NaCl, 10 mM
ZnS04, store at -20°C)
- SOx T AE buffer
- Agarose for gel electrophoresis
- UV transilluminator
- Spectrophotometer
- Ultrafiltration units (cut off molecular weight 30,000 -
100,000, e.g., Ultrafree CL30000LTK, Millipore Co.)
- A programmable thermal controller (An conventional PCR
thermalcycler is available. e.g., Model PTC-ISO, MJ Research)
- PCR tubes fit to the thermal controller
- Additional reagents and equipment for in situ hybridization
with T-T dimer (See Chapter 11)
Procedure
Primer design
A primers pair well conditioned to ordinary symmetric PCR is
probably suitable for asymmetric PCR. Requirements for the
PCR primers, such as equality of melting temperatures, GCI
AT balance, and careful avoidance of primer-dimer formation
are also available in asymmetric PCR. One additional condition
for using T -T dimerization method, is to contain the adjacent
thymines to be sense probe or adjacent adenines to be antisense
probe. If this condition is fulfilled, the primers which were already used to detect the mRNA, are quite suitable for usage.
Template eDNA preparation
eDNA in the 1O- 18 mol of purified cDNA is sufficient for 100 III ofPCR solution.
vector It is recommended to pre-digest it by appropriate restriction enzyme to cut out the cDNA from the vector.
Reverse tran- The first template cDNA for the probe production can be synthescripted eDNA sized by reverse transcription from tissue mRNA. Total RNA is
extracted from the tissue by ordinary guanidium thiocyanate