228
HIDEYO OHUCHI AND SUMIHARE NOTI
Note: When DTT is included in the transcription buffer, add the
same volume of dHzO.
Note: It is not advisable to cut the plasmid DNA with the restriction enzymes that create 3' -protruding ends, such as Apa I, Kpn I,
Pst I, Sac I, and Sph I.
2. Incubate at 37°C for Ihr.
3. Add 2)11 RNA polymerase, 1)11 RNase inhibitor. Incubate at
37°C for Ihr.
4. Chill on ice. Remove a 1)11 aliquot and run on an agarose gel
(1-2%) to estimate the amount synthesized.
Note: This gel must be RNase-free, but does not need to be denaturing. An RNA band lO-fold more intense than the plasmid
band should be seen, indicating that 1 Ollg probe has been synthesized. Use a nucleotide size marker and estimate the size of the
riboprobe synthesized. Unexpected larger size RNA could give
some backgrounds.
5. Add 1)11 RNase inhibitor, 2)11 DNase (RNase-free; Promega,
1 U/)1l). Incubate at 37°C for 15 mins.
6. Add 3)1l4M LiCl, 1)11 yeast tRNA (10mg/ml), 100)11 ethanol
(200-proof). Mix and incubate at -20°C for> Ihr.
7. Spin in amicrofuge for 20 mins at 4°C. Wash pellet with 80 %
ethanol and air-dry the pellet.
8. (Optional; If the transcript is greater than 0.5 kb in length,
reduce the average size to 500 bases). Add an equal volume
(100)11 each) of80 mM NaHC03, 120 mM NazC03 and heat at
60°C for a period of time given by: time (t; min) =(L-Lf)/(k x
Lx Lf), where L is the starting length of the transcript (kb), Lf
is the the desired length (kb), and k is 0.11 (cuts/kb/min).
When Lf is 0.5, t = (L-0.5)/(0.055L).
9. Chill on ice. Add 1)11 glacial acetic acid on ice, mix, add 20)11
3M sodium acetate, 560)11 ethanol. Mix and incubate at -20°C
for >lhr.
10. Spin in amicrofuge for 20 mins at 4°C. Wash pellet with 80%
ethanol, and air-dry the pellet.
HIDEYO OHUCHI AND SUMIHARE NOTI
Note: When DTT is included in the transcription buffer, add the
same volume of dHzO.
Note: It is not advisable to cut the plasmid DNA with the restriction enzymes that create 3' -protruding ends, such as Apa I, Kpn I,
Pst I, Sac I, and Sph I.
2. Incubate at 37°C for Ihr.
3. Add 2)11 RNA polymerase, 1)11 RNase inhibitor. Incubate at
37°C for Ihr.
4. Chill on ice. Remove a 1)11 aliquot and run on an agarose gel
(1-2%) to estimate the amount synthesized.
Note: This gel must be RNase-free, but does not need to be denaturing. An RNA band lO-fold more intense than the plasmid
band should be seen, indicating that 1 Ollg probe has been synthesized. Use a nucleotide size marker and estimate the size of the
riboprobe synthesized. Unexpected larger size RNA could give
some backgrounds.
5. Add 1)11 RNase inhibitor, 2)11 DNase (RNase-free; Promega,
1 U/)1l). Incubate at 37°C for 15 mins.
6. Add 3)1l4M LiCl, 1)11 yeast tRNA (10mg/ml), 100)11 ethanol
(200-proof). Mix and incubate at -20°C for> Ihr.
7. Spin in amicrofuge for 20 mins at 4°C. Wash pellet with 80 %
ethanol and air-dry the pellet.
8. (Optional; If the transcript is greater than 0.5 kb in length,
reduce the average size to 500 bases). Add an equal volume
(100)11 each) of80 mM NaHC03, 120 mM NazC03 and heat at
60°C for a period of time given by: time (t; min) =(L-Lf)/(k x
Lx Lf), where L is the starting length of the transcript (kb), Lf
is the the desired length (kb), and k is 0.11 (cuts/kb/min).
When Lf is 0.5, t = (L-0.5)/(0.055L).
9. Chill on ice. Add 1)11 glacial acetic acid on ice, mix, add 20)11
3M sodium acetate, 560)11 ethanol. Mix and incubate at -20°C
for >lhr.
10. Spin in amicrofuge for 20 mins at 4°C. Wash pellet with 80%
ethanol, and air-dry the pellet.