Rezumatul tezei de doctorat - USAMV Cluj-Napoca

- extension 90 seconds at 68°C
- final extension: 7 minutes at 68°C (after Coalo Maria Chiara, 1999).
The amplification performed on 69 samples, developed on Potato Dextrose Broth
(PDB) medium, using an adapted method after Ristaino and al., 1998.
I.3.8.2. PCR-RFLP techique - Restriction Fragment Length Polymorphism
The amplified fragments were digested with 3 restriction enzymes from Fermentas:
RsaI, HindIII and AluI. The digestion protocol was pursuant to the manufacturer
(http://www.fermentas.com).
The PCR products were overnight incubated, at 37ºC, and at the end at 65ºC for 10
minutes. The digestion products were then separated by electrophoresis, in a 3% agarose gel,
in 1x TBE buffer, for 2 hours at 60V. The DNA was stained with Sybr Safe (Invitrogen), for
the observation of amplified fragments polymorphism, under UV light (Biorad). There was
used a DNA Ladder Low Range (700 bp), for the fragment comparison (Fermentas).
I.3.9. Methods used in establishing the phylogenetic diversity
and relationship of Saprolegniaceae family fungi species
I.3.9.1. Automatic sequencing
The DNA strand was sequenced with universal primers ITS1 (forward) and ITS4
(reverse), by Mycrosinth (Switzerland). The sequencing results were interpreted using a
specific software called Chromas Lite.
For the establishing of genetic diversity there were used specific genetical software.
CHAPTER II
PERSONAL RESEARCH RESULTS
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