HincII (HindII) restriction enzyme

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	Restriction analyses of the three amplification products were performed as described by Hong et al. 12 with four different restriction enzymes, DraI, HincII, HindIII and XbaI (Takara, Japan). Restriction digestion profiles of product A, B or C permitted the determination of the location of restriction sites. When there were three restriction sites in an IGS-PCR product, double-digestion of each product was performed to locate the third restriction site.

Protocol tips

Restriction analyses of the three amplification products were performed as described by Hong et al. 12 with four different restriction enzymes, DraI, HincII, HindIII and XbaI (Takara, Japan). Restriction digestion profiles of product A, B or C permitted the determination of the location of restriction sites. When there were three restriction sites in an IGS-PCR product, double-digestion of each product was performed to locate the third restriction site.

Publication protocol

"Cultures of A. alternata CECT 20560 and 2662 were
obtained from the Spanish type culture collection
(Valencia, Spain) and used as control strains. The
Internal Transcribed Spacer (ITS) of 5.8S and the 26S
regions of nuclear ribosomal DNA were amplified and
sequenced using previously described primers (Fig. 1)
[10,11]. The ITS-5.8S rDNA amplification fragment was also analysed with HaeIII, AluI and HinfI (Takara,
Japan) endonucleases according to de Hoog and
Horre´ [2].
The Intergenic Spacer (IGS) region was amplified
using three combinations of four primers, namely
GVA#30, 26S3111F, IGS27 and CNS1 (Fig. 1) following a previously described method [12] which was
slightly modified for use in these studies. Optimal
annealing temperature was determined to be 628C
and the MgCl2 optimal concentration was found to
be 2 mM. Consequently, the final conditions and PCR
mixture were: 948C 5 min, 35 cycles of 948C 1 min,
628C 1 min, 728C 1 min, and a final step of 728C 7 min;
NH4 Buffer: 16 mM (NH4)2SO4, 67 mM TrisHCl pH
8.8, 0.01%Tween-20; 2 mM MgCl2; 200 mM each
dNTP; 0.4 mM of each primer; 1.25 units of Taq
Polymerase (BioTaq, Bioline); 10 ng of genomic DNA;
25 ml final volume. PCR products were visualized by
1% agarose gel electrophoresis in TBE buffer, EtBr
staining, and UV-illumination. Restriction reactions
were carried out using 1.5 ml of PCR product in 10 ml
reaction mixture for 6 h according to enzyme manufacturer’s recommendations. Digestion products were
resolved in 1% agarose gels in TBE buffer, EtBr
staining and UV-illumination. Restriction analyses of
the three amplification products were performed as
described by Hong et al. [12] with four different
restriction enzymes, DraI, HincII, HindIII and XbaI
(Takara, Japan). Restriction digestion profiles of product A, B or C permitted the determination of the
location of restriction sites. When there were three
restriction sites in an IGS-PCR product, double-digestion of each product was performed to locate the third
restriction sit"

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