Causes Of Errors

Question 1

Low yield or no amplification product?

Possible causes

Answer 1

• low amount of PCR product loaded in electrophoresis
• missed some components
• pipetting errors

• annealing temperature not optimal

• problems in extracted DNA concentration

• problems in PCR reagents or parameters

• suboptimal amplification in general

Question 2

Amplification in the blank
Possible causes

Answer 2

• pipetting carry-over
• contamination in the reagents or materials

Question 3

Not the correct product size?
Possible causes

Answer 3

• mispriming
• primers annealed at a different site

Question 4

Extra bands in electrophoresis
Possible causes

Answer 4

• primers annealed at many sites

• primer is not specific

Question 5

Low yield or
no amplification product?
Possible causes
• low amount of PCR product loaded in electrophoresis
• missed some components
• pipetting errors

Comments

Answer 5

• repeat PCR

Question 6

Low yield or
no amplification product?
Possible causes
• annealing temperature not optimal

Comments

Answer 6

• optimize annealing temperature
• use gradient PCR

Question 7

Low yield or
no amplification product?
Possible causes
• problems in extracted DNA concentration

Comments

Answer 7

• dilute further the DNA for concentrated samples
• or add more DNA for very diluted samples

Question 8

Low yield or
no amplification product?
Possible causes
• problems in PCR reagents or parameters

Comments

Answer 8

• increase one reagent or parameter at a time
• increase DNA
• increase primer
• increase dNTP
• increase MgCl2
• increase Taq pol

Question 9

Low yield or
no amplification product?
Possible causes
• suboptimal amplification in general

Comments

Answer 9

• use additive or enhancing reagents
• add DMSO or betaine – decreases Tm; inhibit secondary
structures in the DNA template or the DNA primers

Question 10

Amplification in the blank
Possible causes
• pipetting carry-over
• contamination in the reagents or materials

Comments

Answer 10

• repeat PCR
• ensure that pipette tips are replaced
• avoid contamination on the reagents

Question 11

Not the correct product size?
Possible causes
• mispriming
• primers annealed at a different site

Comments

Answer 11

• optimize primer design

Question 12

Extra bands in electrophoresis
Possible causes
• primers annealed at many sites

Comments

Answer 12

• too much MgCl2
• too much polymerase
• too much primer

Question 13

Extra bands in electrophoresis
Possible causes
• primer is not specific

Comments

Answer 13

• repeat primer design