Trouble-shooting: Non-specific bands
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Here are some troubleshooting hints that we have gathered
regarding non-specific bands after PCR reactions:
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| Non-optimal Mg++ concentration |
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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 µM of each nucleotide
Suggestions:
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Check the concentration of stock solutions of all
nucleotides
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Double check the final concentrations of all nucleotides
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| DNA contamination / carry-over |
Suggestions:
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Test for carryover by performing PCR without adding
target DNA.
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Avoid carryover (see below)
To prevent carryover, good lab practices should be
used such as:
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Physically isolating PCR preps and products
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Autoclaving solutions, tips, and tubes
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Aliquoting reagents to minimize repeated sampling
(no more than 20 reactions per aliquote)
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Eliminating aerosols by using positive displacement
pipettes
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Premixing reagents
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Adding DNA to reaction last
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Choosing (+) and (-) controls carefully
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Soaking gel box and combs in 1M HCl to depurinate
DNA
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Using new razor blades to exise bands
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Using new razor blades to exise bands
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Covering UV box with fresh plastic wrap
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Always using oil overlay
To eliminate contamination / carryover:
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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
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UNG Digestion: Incorporate d-UTP nucleotides into
reaction and do subsequent uracyl DNA glycosylase digestion
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| Primer annealing temperature is too low
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Primer annealing temperature is typically 50-60°C (may be higher or lower
based on primer sequence and buffer components).
Suggestion: Determine Tm/annealing temperature based on one of
the following equations:
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| 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
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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
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| Mispriming caused by secondary structure
of template, snapback, or excessive homology at 3' ends of primers |
Suggestions:
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Increase initial denaturation temperature to 95-97°C
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Denature DNA minus enzyme & buffer for 4-6 minutes
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Increase cycling denaturation time 15-30 seconds
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Try "Hot Start" technique
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Add T4 Gene 32 protein 3-5 µl/ml
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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
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Consider addition of cosolvent to reaction buffer:
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| Primers are degraded or sequence is not
optimal |
Primers should have same number A & T's versus G &
C's, and they should be at least 14 bases for specificity.
Suggestions:
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If primers are short and A-T rich, add 0.9 - 2.0%(v/v)
DMSO
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If primers are G-C rich, add 1-10% (v/v) Formamide
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Double check priming sequence, use primer design program
if available
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Check aliquot of primers on a gel to ensure they are
not degraded
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| Primer concentration is too high |
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Suggestion: Adjust the primer concentration (0.1 - 1.0 µM of
each primer is optimal)
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| Cycle number is too high |
Most templates require 25-30 cycles.
Suggestion:
Cycle number should be based on starting concentration
of template DNA.
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If
the number of target molecules in your sample is...
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Then
we recommend the following number of Cycles...
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| 3 x 105
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25-30 |
| 1.5 x 104
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30-35 |
| 1.0 x 103
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35-40 |
| 50 |
40-45 |
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| Incorrect template to enzyme ratio
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The necessary amount of template varies from reaction
to reaction. As a guideline, 100 - 750 ng human DNA (105 - 106 copies)
per 100 µl reaction. The amount of enzyme should be optimized for each
template.
Suggestions:
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Titrate the amount of template in the reaction
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Perform optimization experiment varying enzyme concentration
by 0.50 increments in suggested range (0.5 to 5.0 units)
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