Help with nuclease S1 protocol (long!)

[Ned Mantei]

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.
> 
> Thanks
> 
Here is a protocol that has worked well for us:
                                                        J.R. 14.06.90
S1-Mapping                              

Reagents: 

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.

a.) Kinasing:

- 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
  fractions.
- 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
  Biolabs)
- 30' / 37 °C


b.) Filling ends with Klenow:

- Prepurification of the DNA is not necessary; even mini-prep DNA is
  good enough.
- digest DNA with an appropriate restriction enzyme, but don't treat
  with CIAP.
- 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 
possible.


Markers:

- 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.


Hybridization:

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
his Hybrid.Temp.-table!)

- 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).


S1-Reaction:

- 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
  dilution buffer.
- 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 
  buffer.


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
  usual.
- Expose a Kodak X-omatic film at minus 70 °C (preflash if necessary).

-- 
Ned Mantei
Dept. of Neurobiology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland
mantei at neuro.biol.ethz.ch   Fax: +41-1-633-1046