Laboratory Protocol

Question 1

What is the recipe for PCR

Answer 1

• Template DNA
• Forward and Reverse primer (for A and C)
• Water
• TaqPolymerase
• 5 x Taqpolymerase Buffer
• dNTPs
• Magnesium Chloride

Question 2

What do you do when the recipe for PCR is complete?

Answer 2

• Place on ice
• Vortex to mix components
• Centrifuge and collect all components from the bottom
• Place in thermocycler
• 1 Round at 94 degrees for 3 minutes (initial denaturation)
• 35 Cycles of:
• 1 minute 94 (denaturation)
• 1 minute 55 (Annealing)
• 1 minute 72 (Extension)

Question 3

What do you do to run the gel?

Answer 3

• 1% Slab of agarose gel prepared in 1 x TAE buffer (Tris, Acetic acid, EDTA)
• Melted agarose - add ethidium bromide
• Our mixture into gel casting tray and comb to create wells
• Allow gel to set completely
• Load sample onto gel and run for 30 minutes at 70V
• Sample is size factioned
• After running - use UV transilluminator to identify amplified products

Question 4

What size are protocol A products (protist rDNA)

Answer 4

• 528bp

Question 5

What size are Protocol C products (Bonamia Ostreae)

Answer 5

• 780bp

Question 6

What are the advantages of PCR

Answer 6

• Fast ad more accurate identification compared to morphological identification
• allows study of prevalence and distribution of parasite species and strains and efficiently
• can study host-parasite interactions
• can identify parasite factors which influence occurance and severity of the disease
• ability to analyse small amounts
• higher discriminatory power
• can characterise etiological agents

Question 7

What are the disadvantages of PCR

Answer 7

• Requires sufficient DNA sequence information
• Primer design is critical
• primers may interfere with each other leaving some genes and bacteria undetected (She et al., 2010)
• qPCR can give false negatives so to avoid you can run multiple qPCRs in conjunction with each other
• qPRC can only be used to target known genes