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Trouble-shooting: No product

Here are some troubleshooting hints that we have gathered if your PCR yields no product: Non-optimal Mg++ concentration The amount of template in the reaction is not optimal An enzyme inhibitor is present in the reaction Primer annealing temperature is too high or too low Primers are degraded or not optimal Incomplete template denaturation Machine based error Mispriming caused by secondary structure of template, snapback, or excessive homology at 3' ends of primers NaCl concentration above 50 mM KCl concentration above 50 mM Non-optimal Mg++ concentration

Suggestion: Titrate magnesium concentration using our PCR Optimization K it.

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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 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 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 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 Back to Top Primer annealing temperature is too high or 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 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:

  • If primers are short and A-T rich, add 0.9 - 2.0%(v/v) DMSO
  • If primers are G-C rich, add 1-10% (v/v) Formamide
  • Double check priming sequence, use primer design program if available
  • Check aliquot of primers on a gel to ensure they are not degraded
Back to Top Incomplete template denaturat ion 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 94C for 2 minutes should be adequate in most cases. As soon as the sample reaches 94C, 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:
  • 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
  • Closed circular DNA should be cleaved before the PCR, uncut circles renaturate too fast
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 Mispriming caused by secondary structure of template, snapback, or excessive homology at 3' ends of primers 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 NaCl concentration above 50 mM

Suggestion: Reduce NaCl concentration

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KCl concentration above 50 mM

Suggestion: Reduce KCl concentration

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