AIM: The purpose of the exercise is to introduce students to the ribotyping methods. Microorganism differentiation is often based on the genes encoding ribosomal RNA (rRNA) of small and large subunit of the ribosome. These genes, forming the rrn operon, are characteristic for all bacterial organisms. Between the sequences coding the ribosomal subunits 16S, 23S and 5S rRNA, there are polymorphic regions which vary in size and sequence. These regions of the operon are convenient molecular targets used in the phylogenetic studies. The ITS-PCR belongs to the ribotyping methods and it analyses a polymorphic region between the 16S and 23S rRNA coding sequences. The aim of this experiment is to use the ribotyping for the intra- and interspecies differentiation of the supplied bacterial strains (DNA from 6 different species, 3 isolates each).

The kit is designed to perform experiments during two laboratory classes (I – PCR set-up – 1.5-2 hours, II – electrophoresis – 2 hours). We suggest to perform laboratory experiments in six groups of two students.

EasyGenotyping ITS-PCR kit components (1 exercise, DY62):

6 sets of PCR reagents:

• 6 tubes with PCR buffer (10 reactions per tube)
• 6 tubes with 50 mM MgCl₂ solution (10 reactions per tube)
• 6 tubes with ready-to-use premixed dNTPs solution (10 reactions per tube)
• 6 tubes with ready-to-use forward primer L1 solution (10 reactions per tube)
• 6 tubes with ready-to-use reverse primer G1 solution (10 reactions per tube)
• 6 tubes with PCR grade water
• 6 tubes with TaqNova DNA polymerase (10 reactions per tube)
• 18 tubes with genomic DNA isolated from 6 different bacteria species (3 reactions per tube)

Moreover:

• M100-1000 ready-to-use DNA Ladder (10 electrophoretic lanes)
• 6x Green DNA Gel Loading Buffer (0.5 ml)
• 50x TAE buffer for agarose gel electrophoresis (50 ml)
• Agarose (5 g)
• 7 laboratory protocols (one with additional information for a teacher)

EasyGenotyping ITS-PCR kit components (5 exercises, DY625):

6 sets of PCR reagents:

• 6 tubes with PCR buffer (50 reactions per tube)
• 6 tubes with 50 mM MgCl₂ solution (50 reactions per tube)
• 6 tubes with ready-to-use premixed dNTPs solution (50 reactions per tube)
• 6 tubes with ready-to-use forward primer L1 solution (50 reactions per tube)
• 6 tubes with ready-to-use reverse primer G1 solution (50 reactions per tube)
• 6 tubes with PCR grade water
• 6 tubes with TaqNova DNA polymerase (10 reactions per tube)
• 18 tubes with genomic DNA isolated from 6 different bacteria species (15 reactions per tube)

Moreover:

• 7 laboratory protocols (one with additional information for a teacher)
• Agarose (25 g)
• 50x TAE buffer for agarose gel electrophoresis (100 ml)
• M100-1000 ready-to-use DNA Ladder (50 electrophoretic lanes)
• 6x Green DNA Gel Loading Buffer (1.0 ml)