Navigation Links
Trouble-shooting: Smeared bands

Here are some troubleshooting hints that we have gathered regarding smeared bands after PCR reactions: Primer annealing temperature is too low Mg++ concentration is too high Incorrect template to enzyme ratio Nucleotide concentration is too high or unbalanced DNA contamination / carry-over Mispriming caused by excessive homology at 3' ends of primers (primer dimers) DNase activity (indicated by smears visible on gel below expected band size) Oil contamination of gel sample Non-optimal Mg++ concentration

Suggestion: Titrate magnesium concentration using our PCR Optimization Kit.

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 DNA contamination / carry-over Suggestions:
  1. Test for carryover by performing PCR without adding target DNA.
  2. Avoid carryover (see below)
To prevent carryover, good lab practices should be used such as:

  • Physically isolating PCR preps and products
  • Autoclaving solutions, tips, and tubes
  • Aliquoting reagents to minimize repeated sampling (no more than 20 reactions per aliquote)
  • Eliminating aerosols by using positive displacement pipettes
  • Premixing reagents
  • Adding DNA to reaction last
  • Choosing (+) and (-) controls carefully
  • Soaking gel box and combs in 1M HCl to depurinate DNA
  • Using new razor blades to exise bands
  • Using new razor blades to exise bands
  • Covering UV box with fresh plastic wrap
  • Always using oil overlay
To eliminate contamination / carryover:
  • 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
  • UNG Digestion: Incorporate d-UTP nucleotides into reaction and do subsequent uracyl DNA glycosylase digestion
Back to Top Primer annealing temperature is 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 Mispriming caused by secondary structure of template, snapback, or excessive homology at 3' ends of prim ers 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 DNase activity (indicated by smears visible on gel below expected band size)

Suggestion:

  1. Make fresh stock solutions
  2. Check the integrity of template DNA
Back to Top Oil contamination of gel sample

Suggestion:

Spin the reaction tube and carefully extract the oil layer from the surface

Back to Top

Incorrect template to enzyme ratio

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
'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Trouble-shooting: Low yield
2. Trouble-shooting: Non-specific bands
3. Trouble-shooting: Misincorporation or low fidelity
4. Trouble-shooting: No product
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:2/22/2017)... ... February 22, 2017 , ... ... addition of Tom Perkins as European director. Operating from Pennside’s Zurich headquarters, Pennside ... , Perkins joins Pennside after more than a decade with leading market ...
(Date:2/22/2017)... N.C., Feb. 22, 2017  United Therapeutics Corporation (NASDAQ: ... results for the fourth quarter and year ended ... results reflect continued growth as net revenues reached ... Martine Rothblatt, Ph.D., United Therapeutics, Chairman and Chief ... to develop and advance our growing product pipeline, ...
(Date:2/21/2017)... Feb. 21, 2017 /PRNewswire/ - SQI Diagnostics Inc. ("SQI" or the ... results for the three months ended December 31, 2016. ... life sciences and diagnostics company that develops and commercializes proprietary ... ... build on the commercial milestones achieved in fiscal 2016," said ...
(Date:2/21/2017)... ... February 21, 2017 , ... ... Liquid Biopsy System , a fully automated benchtop system for collecting intact circulating ... is being launched at the Molecular Medicine Tri Conference (Tri-Con) Annual Meeting 2017 ...
Breaking Biology Technology:
(Date:2/3/2017)...  Texas Biomedical Research Institute announced that its Board of ... as the Institute,s new President and CEO. Dr. Schlesinger will ... He is currently the Chair of the Department of Microbial ... Interface Biology at Ohio State University. "We are ... CEO of Texas Biomed," said Dr. James O. Rubin ...
(Date:2/2/2017)...  Central to its deep commitment to honor ... Japan Prize Foundation today announced the laureates of ... envelope in their respective fields of Life Sciences ... being recognized with the 2017 Japan Prize for ... to the advancement of science and technology, but ...
(Date:1/26/2017)...  Crossmatch, a leading provider of security and identity ... combatting fraud, waste and abuse in assistance operations around ... on Disaster Relief conference in Panama City ... and foreign assistance organizations throughout Latin America ... a largely unacknowledged problem in the foreign assistance and ...
Breaking Biology News(10 mins):