Trouble-shooting: No product
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| Here are some troubleshooting hints that we have gathered
if your PCR yields no product: |
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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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| The amount of template in the reaction
is not optimal |
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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| An enzyme inhibitor is present in the
reaction |
Suggestion:
Reduce or remove the concentration of any inhibitor in
the reaction mixture. Known inhibitors of PCR include:
50 mM ammonium chloride
EDTA (metal chelator)
> 0.8 µM Hematin
PBS (phosphate will bind free magnesium)
> 0.02 % sarcosyl
0.5 M urea
> 5 % DMF
> 10 % formamide
heparin
> 20 % PEG deoxycholate
> 0.01 % SDS (can be reversed with equal molar ratio of NP40 and Tween
20)
> 10 % DMSO
> 20 % glycerol
> 0.4% N-Octylglucoside
Residual Phenol
>0.06% Sodium deoxycholate
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| Primer annealing temperature is too high
or too low |
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:
| 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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| Primers are degraded or 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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| Incomplete template denaturation |
Insufficient heating during the denaturation step is a
common cause of failure in a PCR reaction. It is very important that the
reaction reaches a temperature at which complete strand separation occurs.
A temperature of about 94°C for 2 minutes should be adequate in most cases.
As soon as the sample reaches 94°C, it can be cooled to
the annealing temperature. Extensive denaturation is probably unnecessary
and limited exposure to elevated temperatures helps maintaining maximum
polymerase activity throughout the reaction.
DNA reaction buffers with higher Mg-concentration (4-5
mM) may require a higher denaturation temperature to allow complete separation
of the DNA template strands. It is recommended to use the supplied buffer
without adding further magnesium.
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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Closed circular DNA should be cleaved before the PCR,
uncut circles renaturate too fast
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| Machine based error |
Suggestions:
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Calibrate the heating block and confirm actual block
temperature
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Run a diagnostic program for the particular machine
- contact the machine manufacturer for specifics
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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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| NaCl concentration above 50 mM |
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Suggestion: Reduce NaCl concentration
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| KCl concentration above 50 mM |
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Suggestion: Reduce KCl concentration
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