Navigation Links
Trouble-shooting: Non-specific bands

Here are some troubleshooting hints that we have gathered regarding non-specific bands after PCR reactions: Non-optimal Mg++ concentration Nucleotide concentration is too high or unbalanced DNA contamination / carryover Primer annealing temperature is too low Mispriming caused by secondary structure of template, snapback, or excessive homology at 3' ends of primers Primers are degraded or sequence is not optimal Primer concentration is too high Cycle number is too high Incorrect template to enzyme ratio 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 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 Primers are degraded or sequence is 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 Primer concentration is too high

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

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 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 6

Related biology technology :

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


(Date:6/27/2016)... 27, 2016  Sequenom, Inc. (NASDAQ: SQNM ... lives through the development of innovative products and services, ... the United States denied its petition to ... of Sequenom,s U.S. Patent No. 6,258,540 (",540 Patent") are ... by the Supreme Court,s Mayo Collaborative Services v. Prometheus ...
(Date:6/27/2016)... ... June 27, 2016 , ... Newly created 4Sight ... solutions to the healthcare market. The company's primary focus is on new product ... marketing strategies that are necessary to help companies efficiently bring their products to ...
(Date:6/27/2016)... --  Ginkgo Bioworks , a leading organism design ... awarded as one of the World Economic Forum,s ... innovative companies. Ginkgo Bioworks is engineering biology to ... in the nutrition, health and consumer goods sectors. ... including Fortune 500 companies to design microbes for ...
(Date:6/24/2016)... ... , ... While the majority of commercial spectrophotometers and fluorometers use the z-dimension ... are higher end machines that use the more unconventional z-dimension of 20mm. Z-dimension ... bottom of the cuvette holder. , FireflySci has developed several Agilent flow cell ...
Breaking Biology Technology:
(Date:6/2/2016)...   The Weather Company , an IBM Business (NYSE: ... capability in which consumers will be able to interact with ... via voice or text and receive relevant information about the ... Marketers have long sought an advertising solution that can create ... relevant and valuable; and can scale across millions of interactions ...
(Date:6/1/2016)... June 1, 2016 Favorable Government ... Administration and Criminal Identification to Boost Global Biometrics System ... released TechSci Research report, " Global Biometrics Market ... Forecast and Opportunities, 2011 - 2021", the global biometrics ... 2021, on account of growing security concerns across various ...
(Date:5/20/2016)...  VoiceIt is excited to announce its new ... By working together, VoiceIt and VoicePass will offer ... take slightly different approaches to voice biometrics, collaboration ... usability. Both ... "This marketing and technology partnership allows ...
Breaking Biology News(10 mins):