Help with nuclease S1 protocol (long!)
mantei at neuro.biol.ethz.ch
Mon Aug 12 09:54:20 EST 1996
In article <320F7A3C.67F at yellow.med.yokohama-cu.ac.jp>,
desai at yellow.med.yokohama-cu.ac.jp wrote:
> Hello Everyone:
> I am looking for a good working protocol of Nuclease S1 assay to
> determine the 5'end of mRNA. I have only double stranded DNA. Can
> anyone help me with good protocol that works with double stranded DNA.
Here is a protocol that has worked well for us:
5 x Kinase-Buffer: 60 mM Tris pH 8, 10 mM MgCl2, 8 mM DTT, 100 mM KCl
FAHB: per ml: 0.907 g Formamide (double crist.) = 0.8 ml
200 ul (2 M NaCl, 200 mM PIPES pH 6.4, 5 mM EDTA)
5 x S1: 1.25 M NaCl, 150 mM NaOAc pH 4.5, 5 mM ZnSO4
S1-Enzyme-Dil.Buffer: 20 mM Tris pH 7.5, 50 mM NaCl, 0.1 mM ZnSO4,
50 % Glycerol, 0.1 mg/ml Gelatine
Gel-loading B.: 90 % (v/v) Formamide, 1 mM EDTA, 0.05 % Bromophenol-
blue, 0.05 % Xylene Cyanol
Getting the labeled fragments
Choose your fragments for labeling in a way that they overhang the 5'
and 3' ends of your RNA for 30 to 50 nucleotides. Otherwise you can't
tell afterwards, whether the S1 has worked or not.
- purify the plasmid or al. on a Sephacryl S-500 column to completely
remove even trace amounts of RNA or short DNA. Works only if the
plasmid is big enough not to become included! (ca. 4 kbp) Check the
fractions by UV through adding an equal vol. of ethidium bromide on
to a glass plate (1 ul is enough). Precipitate the appropriate
- cut the purified plasmid with an appropriate restriction enzyme and
treat with CIAP. To stop the reaction bring to: 7 mM EDTA, 0.2 % SDS
0.3 M NaCl; 15'/65 °C
- phenol- and chloroform-extract, precipitate with EtOH, wash and re-
suspend to ca. 1 ug/ul (if possible)
- for a typical kinasing-reaction use about 2 pmoles plasmid-ends in
ca. 20 ul 1xkinase buffer, containing 50 uCi g-32P-ATP (specific ac-
tivity 5000 Ci/mmol) and 20 u polynucleotide kinase (New England
- 30' / 37 °C
b.) Filling ends with Klenow:
- Prepurification of the DNA is not necessary; even mini-prep DNA is
- digest DNA with an appropriate restriction enzyme, but don't treat
- for a typical reaction use about 2 pmoles ends as well in about 10
to 20 ul of G-50 buffer, containing ca. 8-10 pmoles a-32P-dCTP (40-
50 uCi, 3000 Ci/mmol), 20 uM dNTPs (ex. dCTP) cold and about 40 u/ml
klenow fragment (usually 0.3 ul 5 u/ul to the reaction tube).Stop
the klenow fragment by adding EDTA to 15 mM and by heating to 70 °C
for 5' or phenol extract.
- a.) and b.) are liberated from radioactive mononucleotides by
precipitating them twice with 2.5 M ammoniumacetate:
- add 0.5 vol. 7.5 M ammoniumacetate and 2.5 vol. EtOH. After spin-
ning remove the EtOH, add 20 ul TE, 10 ul 7.5 M ammoniumacetate
and again 2.5 vol. EtOH. Spin, wash, dry and take up in TE.
- if necessary, digest with a second restriction enzyme to remove your
desired fragment from the plasmid. If the volume is not too big, add
ficoll-loading buffer after the digestion directly and separate your
fragments on a LGT agarose gel.
- cut out the bands and recover the DNA from the agarose by phenol ex-
traction. EtOH precipitate and take up in FAHB (about 100 ul).
The higher your specific activity, the better. However, also with only
100'000 cpm Cerenkov/pmol an S1-mapping of a not too rare RNA is still
- If available use different amounts of in vitro RNA in S1 reactions
(1-1000 pg) with your labeled fragments to receive massmarkers.
- if possible digest your labeled fragments with other restriction
enzymes to get labeled DNA fragments of known size. Load about 100
to 500 cpm/band on to the gel.
- also load your labeled 5'- and 3'-fragments untreated on to the gel.
Hybridizationtemperature depends on the GC-content in your fragments.
In the described buffersystem use 50 °C for 40-50 %, 52 °C for 50-
60 % and 54 °C above 60 %. (Maniatis uses an other buffer so don't use
- dry down each RNA-sample together with 10 ug yeast carrier RNA
(careful not to dry completely!)
- take up in 10 ul FAHB containing ca. 0.01 pmol/10 ul of the labeled
fragment(s). Vortex vigorously, heat to 50 °C for 10 ', spin, over-
lay with a drop of paraffinoil and denature for 10 ' at 80 °C.
- immediately transfer to a 50-54 °C waterbath and incubate over night
(at least 10 hours).
- to digest N samples, prepare a diluted S1-solution as follows:
Add (N+1)x44 units of S1-nuclease to ice cold (N+1)x 10 ul S1-enzyme
- prepare (N+1)x 200 ul S1-buffer containing 20 ug/ml single stranded
carrier DNA. Keep on ice. Add the S1-nuclease solution.
- Working one tube at a time add 210 ul of the prewarmed (30°C) S1-Mix
by pipetting under the paraffinoil and mix well by pipetting up and
down several times.
- transfer immediately to a 30 °C waterbath and icubate for 40'.
(if your fragments have EcoRI ends, incubate at 24 °C, so the ends
don't get digested because of partial opening)
- to stop the reaction transfer the mixture to a new tube, containing
200 ul phenol/chloroform 1:1.
- EtOH precipitate the aqueous phase directly (no additional salt),
wash, dry, add 0.8 ul of 0.1 x TE and 3 to 4 ul FA-gel-loading
Electrophoresis and Autoradiography:
- denature the samples at 85 °C for 3', put them on ice and load them
on a 5 or 6 % denaturing (sequencing) polyacrylamide gel, depending
on the length of the fragments you want to see. Whatever plates you
use, make sure you can expose the whole gel between the start and
the bromophenol blue front so you don't loose any information.
- avoid the sharkteeth combs, use regular wide ones!
- if using the shorter IBI-plates apply only 35 watts, on normal
sequencing gels you may go up to 60 watts.
- fix the gel in 10 % MeOH, 10 % acetic acid, papertransfer and dry as
- Expose a Kodak X-omatic film at minus 70 °C (preflash if necessary).
Dept. of Neurobiology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland
mantei at neuro.biol.ethz.ch Fax: +41-1-633-1046
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