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Trouble-shooting: Low yield

Here are some troubleshooting hints that we have gathered regarding low yield after PCR reactions: Primer annealing temperature is too high Template not clean or degraded An enzyme inhibitor is present in the reaction Not enough template in the reaction Extension temperature too high Enzyme activity is low Cycle number is too high Nucleotides hydrolized Nucleotide concentration is too high or unbalanced Primer concentration is too low Machine based error Plateau effect Evaporation Primer annealing temperature is too high

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 Template not clean or degraded

For example, protease contamination can degrade the polymerase.

Suggestions:

  1. Try "Hot start" technique
  2. Purify the DNA template to the highest degree possible, including proteinase K digest
Back to Top An< /b> 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 M 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

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)
Back to Top Extension temperature too high Optimal extension temperature and time is dependent of frament size:
  • 72C for 20 seconds for fragments < 500 bp
  • 72C 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

For Roche Applied Science polymerases, 100% activity is guaranteed through the control date.

Suggestions:

  1. Check the control date. If necessary, obtain fresh enzyme
  2. Try "Hot start" technique to help retain activity through thermal cycling
Back to Top Cycle number is too high

Most templates require 25-30 cycles.

Suggestion:

Cycle number should be based on starting concentration of template DNA.

If the number of target molecules in your sample is...

Then we recommend the following number of Cycles... 3 x 105 25-30 1.5 x 104 30-35 1.0 x 103 35-40 50 40-45 Back to Top Nucleotides hydrolized

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 -20C.

Base
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 C 271 9.1 x 103 U 262 1.0 x 104

The lithium and sodium salts have equivalent st ability 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. Back to Top 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 Primer concentration is too low

Suggestion:

Adjust the primer concentration (0.1 - 1.0 M of each primer is optimal)

Back to Top

Machine based error Suggestions:
  • Calibrate the heating block and confirm actual block temperature
  • Run a diagnostic program for the particular machine - contact the machine manufacturer for specifics
Back to Top Plateau effect Possible causes of the Plateau Effect/solutions:
  1. Utilization of dNTP's - dNTP concentration should be 20-200 M each
  2. End product pyrophosphate inhibition - reduce cycle number to 20-35, to reduce pyrophosphate formation
  3. Incomplete denaturation of product at high concentration - use stepwise cycling, increasing time of denaturation in later cycles
  4. Substrate excess in substrate/enzyme ratio - use stepwise cycling increasing extension time in later cycles or enzyme concentration
Back to Top Evaporation

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 l mineral oil overlay/reaction

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