(none)

Nick Talbot N.J.Talbot at exeter.ac.uk
Tue Apr 25 03:43:22 EST 1995


Dear Sir/Madam.
I am enclosing the text of a handout to a presentation given at the=
 recent
=46ungal Genetics conference in Asilomar, California in a workshop=
 entitled "
Pathogenicity and avirulence genes of plant pathogenic fungi: new=
 and
emerging techniques for their identification and isolation". Prof.=
 Hans van
Etten has urged speakers to deposit this information on the mycology=
 net,
so it is freely available.

Differential cDNA Screening Procedures

        The protocols listed refer to cDNA library construction and
preliminary differential screening procedures.  They are mainly adaptations
of standard procedures as per Sambrook et al. (1989) where theoretical
background information can be found.  Other publications are referred=
 to in
the text.=20
Nick Talbot lab. /John Hamer lab. protocols.  Extra information available=
 from:
N.J.Talbot at cen.exeter.ac.uk

1. cDNA Library Construction
Based upon blast-infected rice leaf cDNA library (Talbot, Ebbole=
 & Hamer-
Plant Cell 5:1575-90, 1993).  RNA extractions were carried out by=
 a
Guanidinium Isothiocyanate procedure (Sambrook et al. 1989) and poly(A)+
purified over Oligo(dT) columns.  The library was constructed in=
 lGEM4
(Promega).

(a) ANNEALING REACTION

1.  Resuspend Xba-oligo(dT) primer adaptor ( or an oligo-dT primer)=
 in
sterile water, at a concentration of 1 =B5g/=B5l.
2.  Combine the following on ice.

Poly(A+) RNA from healthy rice leaves   10 ul (10=B5g poly(A)+RNA)
3.0 M NaOAc             1.0 ul
Ethanol         25 ul

Chill at -20oC, spin down RNA pellet.  Dry down in a speed vac dessicator.
3. Resuspend in 10 =B5l of primer.
4. Heat to 65oC for 5.0 min.=20
5.  Quick chill in ethanol ice bath.

(b) FIRST-STRAND SYNTHESIS (After Brown and Kafatos, 1988:  J. Mol.=
 Biol.
203:425-437):

1.  To the annealed oligo and poly(A)+ RNA add the following on ice:

1.25X RT Buffer (-CTP)  80 =B5l
5-methyl dCTP   10 =B5l
RNasin (Promega)        1.0 =B5l
M-MLV RT (BRL)  2.0 =B5l

1.25X RT Buffer (-CTP) is:
1.0M Tris-HCl (pH 8.7)  100 =B5l  100mM
100 mM DTT      10 =B5l   10 mM
1.0M KCl        40 =B5l   40 mM
1.0 MgCl2       8 =B5l    8 mM
100 mM dGTP (Pharmacia) 8 =B5l    0.8 mM
100 mMdATP      8 =B5l
100 mM TTP      8 =B5l
80 mM Na4P2O7   5 =B5l    4.0 mM
(500 =B5g/ml) actinomycin D       100 =B5l  50 =B5g  =20
H20     597 =B5l

Mix well and dispense in 100 =B5l aliquots and store at -70oC.
2.  Incubate the reaction at 40oC for 1.0 hour.
3. Terminate reaction by placing on ice and extract with 100 =B5l=
 of
phenol/chloroform.  Remove 95 =B5l of aqueous and reextract organic=
 phase
with another 100 =B5l of 0.1X sterile TE.
4.  Precipitate the DNA:RNA hybrid with 0.1 vol. sodium acetate (20=
 =B5l) and
two volumes (440 =B5l) of ethanol.  Chill in an ice bucket for 10=
 min. and
pellet by centrifugation for 20 minutes in the cold.
5.  Drain ethanol and resuspend pellet in 20 =B5l of TE and precipitate=
 with
ammonium acetate  (20 =B5l of ammonium acetate, 80 =B5l of ethanol).=
  Allow to
stand at least 2-3 hrs (O/N is better according to BRL).  Spin down=
 at room
temperature.  Wash pellet in 70% ethanol.
6.  Dry in the Speed Vac and resuspend in 10 =B5l of 0.1X TE .

(c) SECOND STRAND SYNTHESIS (Polites and Marrotti, Biotechniques=
 1986 4:
514-520).

1. To the dried sscDNA pellet, add the following:
1 st strand cDNA        10.0 =B5l
0.4M Hepes pH 7.6       25.0 =B5l
60mM MgCl2      10.0 =B5l
1.0 M DTT       1.0 =B5l
1.0M KCl        6.0 =B5l
5.0mM dNTP mix  10.0 =B5l
10 mM =DF-NAD     1.5 =B5l
P32-dCTP        10 =B5l
DNA Pol 1       15.0=B5l
RNase H 3.0 =B5l
E. coli ligase  1.0 =B5l
dH2O    7.5 =B5l

2. Mix well on ice and incubate at 14oC for 1.0 hr. then shift to=
 RT for an
additional hour.
3. Stop the reaction by placing on ice.  Perform one phenol/chloroform
extractions (reextracting the organic layer) and precipitate once=
 with
sodium acetate and ethanol.  Dry the pellet and resuspend in 5 ul=
 of 0.1X
TE.

(d) BLUNT ENDING THE cDNA

1. To the cDNA add the following:

10X NTB (Maniatis nick-translation buffer)              3 =B5l
5mM dNTPs                       1 =B5l
H20                     20 =B5l
T4 DNA polymerase                       0.5 =B5l
2.  Incubate at 37 C for 20 min.  Adjust aqueous volume to 100 =B5l=
 (by
adding 70 =B5l of 0.1XTE).  Perform one phenol chloroform extraction,
reextracting the aqueous volume and precipitate the DNA by ammonium=
 acetate
precipitation.  Wash pellet with 70% ethanol and dry in speed vac.=
=20
Resuspend cDNA in 10 =B5l of 0.1XTE.

(e) ADDING LINKERS
1. We prepare and test linkers as in Sambrook et al. (1989).

Combine the following on ice.

double -stranded cDNA   10.0 =B5l
10X Ligation buffer     2.0 =B5l
Kinased linkers 5.0 =B5l
dH20    2.0 =B5l
10mM ATP        0.5 =B5l
T4-DNA ligase   0.5 =B5l

Mix well and incubate at 14oC overnight.  Check all centrifuges and=
 work
area for radioactivity.=20
(f.) SIZE FRACTIONATION OF cDNA AND LIGATION TO LAMBDA.
1. Save 0.5 =B5l of the ligation mix in 5 =B5l of TE with bromphenol=
 blue stop dye.
2. Heat inactivate ligase by heating at 65 C for 10 min.
3. To the ligation mix add:

10X Restriction buffer  10.0 =B5l
10X Gelatin (1mgml-1)   10.0 =B5l
dH2O    56.0 =B5l
XbaI    2.0 =B5l

Incubate for 1.0 hr at 37oC
Add 2.0 =B5l of EcoRI and continue incubation for 2 more hours. =
 During this
time set-up a 5% acrylamide gel.  (Note: This step for directional=
 Eco-Xba
cDNA cloning)
3. Remove restriction digest to ice and store at -20 C.  Remove 3.0=
 =B5l for
gel analysis.  Combine with 5 ul of TE with bromphenol blue stop=
 dye.
4. Electrophorese on a 5% acrylamide gel, cDNA samples before and=
 after
digestion with restriction enzymes.  Include size standards.  Dry=
 gel and
expose overnight to film.
5. Set-up Biorad agarose A-50M column.  Phenol/chloroform extract=
 cDNA to
remove restriction enzymes.  Reextract organic phase.  Precipitate=
 with
NaOAc and ethanol.  Spin drain pellet and resuspend in 0.1X TE.
6.  Load on to column pre-equilabrated with 0.1X TE.  Collect 200=
 =B5l
fractions in eppendorf tubes (approximately 40).  Monitor radioactivity.=
=20
Assay 2.0 =B5l of every second fraction on a 1.0% TAE agarose gel=
 if you have
doubts.  The separation of cDNA from kinased linkers should be obvious!!
7. Dry gel and expose to film.  Pool fractions with cDNA equal to=
 or
greater than 500 bp (if you want).  Concentrate by butanol extraction=
 and
ammonium acetate precipitate.
8.  To the dried cDNA pellet add:

dH20    6.0 =B5l
10X ligation buffer     1.0 =B5l
Lambdagem4 DNA* 2.0 =B5l
10mM ATP        0.5 =B5l
T4 Ligase       0.5 =B5l

Incubate at 10-15oC overnight, continue at RT the next day.

9.  Packaging follows next day.
* Prepping the Lambda vector before cloning.

1.  We prepare 25 =B5g of lambda vector.  The Lambda DNA is (25ug/100=
 =B5l of
0.1XTE) heated to 68 C for 10 min. and allowed to cool slowly at=
 room temp.
 The cos- annealed Lambda DNA is then ligated in a total volume 200=
 =B5l
overnight.
2.  The cos-ligated lambda DNA is then adjusted to 300 =B5l with=
 10XRE, and
dH20 and digested with Eco and Xba overnight.
3.  The enzymes are heat inactivated, and an appropriate amount of=
 CIAP is
added (we calculate the required amount according to the formulas=
 given in
the product sheet from Boehringer).
4.  After phosphatase treatment at 37oC for 45 min. we inactivate=
 with a
1/10 th volume of EGTA, heat at 70oC for 45 min., and do 2
phenol/chloroform extractions, and ethanol ppt. the lambda DNA.
5. We repeat, enzyme digestion and phosphatasing a second time. =
 The final
Lambda DNA is resuspended in 20 =B5l.  Very low backgrounds of
nonrecombinants are obtained (6 pFu/ug of vector packaged).=20
6. Packaging by standard procedures using Stratagene packaging extracts.

=0C(f.) Differential cDNA Screen

1. Plate out at library at high density using standard procedures=
 (Sambrook
et al. 1989).  Allow plaques to become visible but not to large (5-6h=
 at
37oC).
2. Chill plates for at least two hours before carrying out plaques=
 lifts.
3. Carry out duplicate plaques lifts.  First lift gets 30 sec contact=
 with
plate, second lift 2 min.
4. Incubate nitrocellulose discs in denaturation buffer 30 sec, then
neutralisation buffer for 30 sec.  Standard recipes.
5. Wash filters in 2 x SSPE, rubbing off any adhering agar (This=
 doesn't
affect plaque blots).
6.  Air dry the filters, then bake at 80oC for two hours under vacuum=
 (Call
me a traditionalist).
7. Prehybridise o/n, Then hybridise with + and - cDNA probes.

8. First lifts always get probed with the - probe (in this example=
 healthy
rice leaf cDNA).  Second lifts get the + probe (Infected rice leaf=
 cDNA).
cDNA probes are made by carrying out a first strand cDNA reaction=
 with 1mg
Poly(A+) RNA as per the recipe above.  The reaction is incubated=
 for 1h.
The probe is purified through a Biogel p60 column and denatured with
boiling or NaOH treatment before use.  This is essentially standard=
 (kits
available), but simply follow the first steps of the library protocol=
 if in
doubt.
9. Hybridise 12h.  You can also do a 48h hybridisation to screen=
 for low
abundance cDNAs.
10. Autoradiograph as usual.
11.  Carry out secondary screens in the same way.  Phage DNA can=
 be
extracted conventionally or use single plaque PCR, or PCR from a=
 plaque
pure SM  -70oC stock to get the cDNA insert.  Use T7/SP6 primers=
 for GEM4,
or T3/T7 for ZAP.  Sequencing can be direct based on solid phase=
 protocols,
or conventional dsDNA or ssDNA protocols.
12. Southern blot your clones against genomic DNA sooner rather than=
 later.


Any problems with the above, and I am happy to offer advice by E-mail.=
=20
Much of the above can be supplied in kit forms by major suppliers,
particularly Stratagene, Promega and BRL. Their tech. service are=
 normally
pretty helpful.

=20

__________________________________________________________________________
Dr Nicholas J Talbot,                           Tel 44 1392 264673
Lecturer,                                       Fax.44 1392 264668
Dept. of Biological Sciences,
University of Exeter,
Washington Singer Labs.,
Perry Road, Exeter EX4 4QG, UK.






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