Table. PCR Troubleshooting
Trouble : No PCR products
|
Cause
|
Solution
|
Template DNA that could not be
amplified
(e.g., Template DNA with a high GC
content)
|
Add DMSO (2-5%) and reduce the enzyme
concentration to 0.5 unit.
Use other organic solvents that reduce
Tm.
|
Problems with template DNA.
|
Examine concentration, quality and
purity of template DNA.
Confirm that template DNA is not
degraded by agarose gel analysis.
Carry out test PCR with a pair of
other primers that were successful.
Prepare a new template DNA.
|
Low concentrations of polymerase
|
Increase the amount of enzyme
concentration by 0.5 unit.
If necessary, increase the amount of
enzyme up to 5 units in 100-μl reaction.
|
Low concentrations of MgCl2
|
Increase MgCl2 concentration
by 0.25 mM.
|
Inadequate PCR cycles
|
Reduce annealing temperature.
Increase the number of PCR cycles.
Check whether the final elongation
step (72℃, 5 min) is carried out.
|
Unsuitable primers
|
Prepare a new pair of primers.
|
Inadequate primer concentration.
|
Use of equal concentration of two
primers.
Titrate primers for their optimal
concentrations (from 0.1 to 0.6 μM).
|
Primer quality and storage
|
Confirm that the primers are not
degraded.
Store primers at -15℃ to -30℃.
|
Formation of primer dimers
|
Carry out hot-start PCR
Divide the PCR mixture into two
submixtures, each of which is inactive, and
combine them together just before PCR
reaction.
|
Trouble : Multiple or smeared PCR
products
|
Cause
|
Solution
|
Low annealing temperature.
|
Increase the annealing temperature
based upon the length of primers and their
nucleotide sequences.
|
Low concentration of primers or
inappropriate primers
|
Check whether primers are optimally
designed.
Determine the optimal primer
concentration by titration between 0.1 to 0.6 mM.
Use the same concentrations of the two
primers
Carry out nested PCR*
|
Template DNA that could not be
amplified
(e.g., Template DNA with a high GC
content)
|
Add DMSO (2-5%) and reduce the enzyme
concentration to 0.5 unit.
Use other organic solvents that reduce
Tm.
|
Problem with DNA template
|
Use template DNA after serial
dilution.
|
* Nested PCR : A PCR that uses the
amplified products obtained from primary PCR as template and another pair of primers that are designed
to anneal within the amplified product.
| |
IP : 118.217.216.***
|