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Trouble-shooting: Smeared bands

Here are some troubleshooting hints that we have gathered regarding smeared bands after PCR reactions: Primer annealing temperature is too low Mg++ concentration is too high Incorrect template to enzyme ratio Nucleotide concentration is too high or unbalanced DNA contamination / carry-over Mispriming caused by excessive homology at 3' ends of primers (primer dimers) DNase activity (indicated by smears visible on gel below expected band size) Oil contamination of gel sample Non-optimal Mg++ concentration

Suggestion: Titrate magnesium concentration using our PCR Optimization Kit.

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Nucleotide concentration is too high or unbalanced

The standard concentration is 20-200 M of each nucleotide

Suggestions:

  1. Check the concentration of stock solutions of all nucleotides
  2. Double check the final concentrations of all nucleotides
Back to Top DNA contamination / carry-over Suggestions:
  1. Test for carryover by performing PCR without adding target DNA.
  2. Avoid carryover (see below)
To prevent carryover, good lab practices should be used such as:

  • Physically isolating PCR preps and products
  • Autoclaving solutions, tips, and tubes
  • Aliquoting reagents to minimize repeated sampling (no more than 20 reactions per aliquote)
  • Eliminating aerosols by using positive displacement pipettes
  • Premixing reagents
  • Adding DNA to reaction last
  • Choosing (+) and (-) controls carefully
  • Soaking gel box and combs in 1M HCl to depurinate DNA
  • Using new razor blades to exise bands
  • Using new razor blades to exise bands
  • Covering UV box with fresh plastic wrap
  • Always using oil overlay
To eliminate contamination / carryover:
  • UV irradiation: Mix all components, except template DNA, irradiate in clear 0.5 ml polypropylene tubes in direct contact with glass transilluminator (254 and 300 nm UV bulbs) for 5 minutes
  • UNG Digestion: Incorporate d-UTP nucleotides into reaction and do subsequent uracyl DNA glycosylase digestion
Back to Top Primer annealing temperature is too low

Primer annealing temperature is typically 50-60C (may be higher or lower based on primer sequence and buffer components).

Suggestion:

Determine Tm/annealing temperature based on one of the following equations:

If primers are 20-35 bases If primers are 14 - 70 bases Tp = 22 + 1.46(Ln)
Ln = 2(# G or C) + (# A or T)
TP = Effective annealing
temperature 2 - 5 Tm = 81.5 + 16.6 (log10 [J+]) + 0.41
(% G + C) - (600/l) - 0.063
(% Formamide) + 3 to 12
[J+] = concentration of Monovalent cations
l = length of oligo Back to Top Mispriming caused by secondary structure of template, snapback, or excessive homology at 3' ends of prim ers Suggestions:
  • Increase initial denaturation temperature to 95-97C
  • Denature DNA minus enzyme & buffer for 4-6 minutes
  • Increase cycling denaturation time 15-30 seconds
  • Try "Hot Start" technique
  • Add T4 Gene 32 protein 3-5 l/ml
  • When designing primers, make sure there is no more than 2 bases of homology at the 3' end. Use a primer design program if available
  • Consider addition of cosolvent to reaction buffer:
  • 3-15% DMSO
  • 1-10% Formamide
  • 5-15% Polyethylene glycol
  • 10-15% glycerol
Back to Top DNase activity (indicated by smears visible on gel below expected band size)

Suggestion:

  1. Make fresh stock solutions
  2. Check the integrity of template DNA
Back to Top Oil contamination of gel sample

Suggestion:

Spin the reaction tube and carefully extract the oil layer from the surface

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Incorrect template to enzyme ratio

The necessary amount of template varies from reaction to reaction. As a guideline, 100 - 750 ng human DNA (105 - 106 copies) per 100 l reaction. The amount of enzyme should be optimized for each template.

Suggestions:

  1. Titrate the amount of template in the reaction
  2. Perform optimization experiment varying enzyme concentration by 0.50 increments in suggested range (0.5 to 5.0 units)
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1. Trouble-shooting: Low yield
2. Trouble-shooting: Non-specific bands
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4. Trouble-shooting: No product
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