PCR Protocol Materials Required Flashcards
Preparation
Step 1
• Thaw on ice all PCR components shown in the table. (7)
• All components should be completely thawed to ensure correct concentrations are pipetted.
Nuclease-free water
Taq buffer dNTP
MgCh
Forward primer
Reverse primer
Taq pol
DNA
Step 2
• Properly___ tubes
label
Step 3
• Prepare a _____following the table on the right.
• Be sure to add____ first and
_____last.
PCR cocktail (master mix)
water
DNA sample
Step 4
• Mix the PCR cocktail by gentle inversion and pool contents by a quick run in the centrifuge.
• Pipet out 13 uL of the cocktail to each tube.
• Add 2 uL of the DNA sample in each tube.
• for negative control (blank), use_____ instead of DNA sample
• for positive control, use_____
nuclease-free water
known DNA
Step 5
• Switch on the____ and set program based on the profile shown (next slide).
• The program will last for around____
thermal cycler
2 hours.
Step 6
• Visualize the PCR products using the_____
• Store PCR products at_____ if not used immediately.
agarose gel electrophoresis method
-20°C
Low yield or no amplification product?
Possible causes
• low amount of PCR product loaded in electrophoresis
• missed some components
• pipetting errors
Comments
(1)
• repeat PCR
Low yield or no amplification product?
Possible causes
• annealing temperature not optimal
Comments
(2)
• optimize annealing temperature
• use gradient PCR
Low yield or
no amplification product?
Possible causes
• problems in extracted DNA concentration
Comments
(2)
• dilute further the DNA for concentrated samples
• or add more DNA for very diluted samples
Low yield or
no amplification product?
Possible causes
• problems in PCR reagents or parameters
Comments
(5)
• increase one reagent or parameter at a time
• increase DNA
• increase primer
• increase dNTP
• increase MgCl2
• increase Taq pol
Low yield or
no amplification product?
Possible causes
• suboptimal amplification in general
Comments
(1)
• use additive or enhancing reagents
• add DMSO or betaine – decreases Tm; inhibit secondary
structures in the DNA template or the DNA primers
Amplification in the blank
Possible causes
• pipetting carry-over
• contamination in the reagents or materials
Comments
(3)
• repeat PCR
• ensure that pipette tips are replaced
• avoid contamination on the reagents
Not the correct product size?
Possible causes
• mispriming
• primers annealed at a different site
Comments
(1)
• optimize primer design
Extra bands in electrophoresis
Possible causes
• primers annealed at many sites
Comments
(3)
• too much MgCl2
• too much polymerase
• too much primer
Extra bands in electrophoresis
Possible causes
• primer is not specific
Comments
(1)
• repeat primer design
AVOID CONTAMINATION
• The reagents for PCR should be prepared separately and
used solely for this purpose.
•_______ of all solutions, except dNTPs, primers and Taq DNA Polymerase is recommended.
• Solutions should be aliquoted in small portions and stored in designated PCR areas.____ should be stored separately from other DNA samples.
• A control reaction, omiting template DNA, should always
be performed, to confirm the absence of contamination.
Autoclaving
Aliquots
