FastDigest TasI

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The DNA material was incubated with FastDigest® Tsp509I (TasI) restriction enzyme (Fermentas International, Inc.) at 65˚C for 1 h.	DNA fragments of 135, 73, 70, 60 and 19 bp, were obtained as a set of representative, typical (wild-type) alleles, whereas segments of 135, 92 (73+19 bp), 70 and 60 bp, constituted the set of polymorphic alleles (33). RFLP products were separated by electrophoresis on an 8% polyacrylamide gel, stained with ethidium bromide and observed in UV light. Representative, typical homozygotes, as well as heterozygotes, were sequenced and used as internal control.

Protocol tips
The DNA material was incubated with FastDigest® Tsp509I (TasI) restriction enzyme (Fermentas International, Inc.) at 65˚C for 1 h.
Downstream tips
DNA fragments of 135, 73, 70, 60 and 19 bp, were obtained as a set of representative, typical (wild-type) alleles, whereas segments of 135, 92 (73+19 bp), 70 and 60 bp, constituted the set of polymorphic alleles (33). RFLP products were separated by electrophoresis on an 8% polyacrylamide gel, stained with ethidium bromide and observed in UV light. Representative, typical homozygotes, as well as heterozygotes, were sequenced and used as internal control.

Publication protocol

Exponential amplification of DNA segments for the N363S polymorphism was carried out using a forward primer (5'-CCA GTA ATG TAA CAC TGC CCC-3') and a reverse primer (5'-TTC GAC CAG GGG AAG TTC AGA-3') according to a standard PCR protocol (33). Starter binding to complementary DNA matrix sites was conducted at 56˚C. Amplified DNA sequences of 357 bp length were obtained. The material was incubated with FastDigest® Tsp509I (TasI) restriction enzyme (Fermentas International, Inc.) at 65˚C for 1 h (33). DNA fragments of 135, 73, 70, 60 and 19 bp, were obtained as a set of representative, typical (wild-type) alleles, whereas segments of 135, 92 (73+19 bp), 70 and 60 bp, constituted the set of polymorphic alleles (33). RFLP products were separated by electrophoresis on an 8% polyacrylamide gel, stained with ethidium bromide and observed in UV light. Representative, typical homozygotes, as well as heterozygotes were sequenced and used as internal contro

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