AIM: The purpose of the exercise is to carry out a genomic DNA isolation from buccal swabs and a PCR reaction with the isolated DNA templates in order to detect the mutation in the ccr5 gene which confers resistance to HIV in humans. The detection is based on an amplification of ccr5gene fragments:

• 220 bp in homozygotes without the mutation
• 188 bp in homozygotes with the mutation
• 220 and 188 bp in heterozygote with the mutation

The kit is designed to perform experiments during two or three laboratory classes (I – DNA extraction and PCR set-up – 2-2.5 hours; II – electrophoresis – 1.5-2 hours or I – DNA extraction – 1-1.5 hours; II – PCR set-up – 1-1.5 hours; III – electrophoresis – 1.5-2 hours). We suggest to perform laboratory experiments in six groups of two students.

EasyPCR HIV kit components (1 exercise, DY25)

6 sets of reagents for  DNA isolation from buccal swabs
(each set for 4 isolations)
6 sets of reagents for PCR:

• 6 tubes with 10x reaction buffer (10 reactions per tube)
• 6 tubes with MgCl₂ solution (10 reactions per tube)
• 6 tubes with ready-to-use dNTPs mixture (10 reactions per tube)
• 6 tubes with FORWARD primer (10 reactions per tube)
• 6 tubes with REVERSE primer (10 reactions per tube)
• 6 tubes with PCR – grade water
• 6 tubes with thermostable DNA polymerase (10 reactions per tube)
• 6 tubes with Positive control gene: 220 bp fragment (5 reactions per tube)
• 6 tubes with Positive control gene: 188 bp fragment (5 reactions per tube)

Moreover:

• 7 laboratory protocols (one with additional information for a teacher)
• Agarose (5 g)
• 50x TAE buffer for agarose gel electrophoresis (50 ml)
• Ready-to-use M100-500 DNA Ladder for 10 electrophoretic lanes
• 6x GREEN DNA Gel Loading Buffer (0.5 ml)

EasyPCR HIV kit components (5 exercises, DY255):

6 sets of reagents for  DNA isolation from buccal swabs
(each set for 20 isolations)
6 sets of reagents for PCR:

• 6 tubes with 10x reaction buffer (50 reactions per tube)
• 6 tubes with MgCl₂ solution (50 reactions per tube)
• 6 tubes with ready-to-use dNTPs mixture (50 reactions per tube)
• 6 tubes with FORWARD primer (50 reactions per tube)
• 6 tubes with REVERSE primer (50 reactions per tube)
• 6 tubes with PCR – grade water
• 6 tubes with thermostable DNA polymerase (50 reactions per tube)
• 6 tubes with Positive control gene: 220 bp fragment (25 reactions per tube)
• 6 tubes with Positive control gene: 188 bp fragment (25 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)
• Ready-to-use M100-500 DNA Ladder for 50 electrophoretic lanes
• 6x GREEN DNA Gel Loading Buffer (1 m)