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Troubleshooting of PCR reactions (ver. Eng)

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.

 

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