124
PETER IGARASHI
tor with Geiger counter). Dry pelletunder vacuum, and resuspend in 5 J.ll
DEPC-treated water.
f. Add 10 J.ll ofloading buffer, and run the entire sample on the gel (heat at
85°C for 5 minutes and place on ice before loading). Electrophorese at 60
W for at least 1.5 hours.
g. Separate plates and cover the gel with plastic wrap. Expose to X-ray
film for 30-60 seconds. Locate the riboprobe on the autoradiogram and
excise the corresponding gel band with a razor blade.
h. Elute the gel slice in 400 J.ll of elution buffer at 37°C for 4 hours with
continuous agitation. Remove the eluate to a new microcentrifuge tube.
Monitor the eluate and the gel slice with a Geigercounter (there should
be more counts in the eluate).
i. Add 1 ml of ice-cold ethanol and place on ice for 15 minutes.
j. Centrifuge at 10,000x g for 15 minutes. Allcounts should precipitate
with ethanol. Re-dissolve the RNA pellet in DEPC-treated water and
count 1 J.ll in a liquid scintillation counter.
Comments
• For linearization of plasmid, it is important to select a restriction endonuclease that generates a blunt or protruding 5' end since recessed 5'
ends cause aberrant initiation of transcription. Alternatively, recessed
5' ends can be repaired by treatment with T4 DNA polymerase in the
presence of excess dNTPs (Ausubel et al. 1993).
• Do not place the in vitro transcription reaction mixture on ice; spermidine in the buffer may precipitate the template DNA.
• Although digestion with deoxyribonuclease is sufflcient for preparation
of riboprobe for hybridization experiments, we have found that for ribonuclease protection assays, complete removal of template DNA by
polyacrylamide gel electrophoresis is required.
• The specific activity of the riboprobe can be adjusted by altering the ratio
oflabeled and unlabeled UTP, although the total concentration should
remain greater than 20 J.lM to avoid premature termination.
PETER IGARASHI
tor with Geiger counter). Dry pelletunder vacuum, and resuspend in 5 J.ll
DEPC-treated water.
f. Add 10 J.ll ofloading buffer, and run the entire sample on the gel (heat at
85°C for 5 minutes and place on ice before loading). Electrophorese at 60
W for at least 1.5 hours.
g. Separate plates and cover the gel with plastic wrap. Expose to X-ray
film for 30-60 seconds. Locate the riboprobe on the autoradiogram and
excise the corresponding gel band with a razor blade.
h. Elute the gel slice in 400 J.ll of elution buffer at 37°C for 4 hours with
continuous agitation. Remove the eluate to a new microcentrifuge tube.
Monitor the eluate and the gel slice with a Geigercounter (there should
be more counts in the eluate).
i. Add 1 ml of ice-cold ethanol and place on ice for 15 minutes.
j. Centrifuge at 10,000x g for 15 minutes. Allcounts should precipitate
with ethanol. Re-dissolve the RNA pellet in DEPC-treated water and
count 1 J.ll in a liquid scintillation counter.
Comments
• For linearization of plasmid, it is important to select a restriction endonuclease that generates a blunt or protruding 5' end since recessed 5'
ends cause aberrant initiation of transcription. Alternatively, recessed
5' ends can be repaired by treatment with T4 DNA polymerase in the
presence of excess dNTPs (Ausubel et al. 1993).
• Do not place the in vitro transcription reaction mixture on ice; spermidine in the buffer may precipitate the template DNA.
• Although digestion with deoxyribonuclease is sufflcient for preparation
of riboprobe for hybridization experiments, we have found that for ribonuclease protection assays, complete removal of template DNA by
polyacrylamide gel electrophoresis is required.
• The specific activity of the riboprobe can be adjusted by altering the ratio
oflabeled and unlabeled UTP, although the total concentration should
remain greater than 20 J.lM to avoid premature termination.