Trouble-shooting: Low yield
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Here are some troubleshooting hints that we have gathered
regarding low yield after PCR reactions:
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| Primer annealing temperature is too high
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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
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If primers are 14 - 70 bases
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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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| Template not clean or degraded |
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For example, protease contamination can degrade the polymerase.
Suggestions:
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Try "Hot start" technique
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Purify the DNA template to the highest degree possible,
including proteinase K digest
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| An enzyme inhibitor is present in the
reaction |
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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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| Not enough template in the reaction |
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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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| Extension temperature too high |
Optimal extension temperature and time is dependent of
frament size:
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72°C for 20 seconds for fragments < 500 bp
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72°C for 40 seconds for fragments 1.2 bp.
Suggestion:
Longer times, not higher temperatures should be used when longer templates
or suspected secondary structure is present.
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| Enzyme activity is low |
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For Roche Applied Science polymerases, 100% activity is guaranteed through
the control date.
Suggestions:
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Check the control date. If necessary, obtain fresh
enzyme
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Try "Hot start" technique to help retain activity
through thermal cycling
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| Cycle number is too high |
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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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| Nucleotides hydrolized |
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Suggestions:
Always store nucleotide stock solutions at a concentration of at least
10 mM, 100 mM is best. Significant hydrolysis occurs after storing at
1 mM for 2 months. Dissolve NTPs or dNTPs in water at an expected concentration
of 10 mM. Using 0.05 M Tris-base and pH paper adjust the pH to 7.0. Dilute
an aliquote of the neutralized NTP or dNTP appropriately and read the
optical density at the wave lengths given in the following table. Calculate
the actual concentration using the values for the extinction coefficients.
Freeze away in small aliquots at -20°C.
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Base
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Wavelength |
Extinction coefficients for bases
e(M-1 cm-1) |
| A |
259 |
1.54 x 104 |
| T |
260 |
7.4 x 103 |
| G |
253 |
1.37 x 104
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| C |
271 |
9.1 x 103 |
| U |
262 |
1.0 x 104 |
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| The lithium and sodium salts have equivalent stability and work equally
well in PCR, sequencing, and labeling applications. Lithium salts are more
soluble in ethanol than sodium salts. Thus, removal of lithium salts by
ethanol precipitation is more efficient than removal of sodium salts. Using
lithium salt nucleotide preparations reduce salt-induced artifacts and increase
the legibility of sequencing gels. |
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| Nucleotide concentration is too high or
unbalanced |
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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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| Primer concentration is too low |
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Suggestion:
Adjust the primer concentration (0.1 - 1.0 µM of each primer is optimal)
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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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| Plateau effect |
Possible causes of the Plateau Effect/solutions:
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Utilization of dNTP's - dNTP concentration should
be 20-200 µM each
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End product pyrophosphate inhibition - reduce cycle
number to 20-35, to reduce pyrophosphate formation
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Incomplete denaturation of product at high concentration
- use stepwise cycling, increasing time of denaturation in later cycles
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Substrate excess in substrate/enzyme ratio - use stepwise
cycling increasing extension time in later cycles or enzyme concentration
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| Evaporation |
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Evaporation can lead to higher concentrations of components, which may
inhibit enzyme activity. The change in volume also leads to changes in
the thermal profile inside the reaction tubes.
Suggestion: Use 100 µl mineral oil overlay/reaction
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