Navigation Links
Trouble-shooting: Misincorporation or low fidelity

Here are some troubleshooting hints that we have gathered regarding misincorporation or low fidelity of PCR reactions: Magnesium concentration too high Nucleotide concentration is too high or unbalanced The pH of the reaction buffer is too high Mispriming caused by secondary structure of template, snapback, or excessive homology at 3' ends of primers Damaged template DNA 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 The pH of the reaction buffer is too high A pH 8.3 is optimal Suggestions:
  1. pH of reaction buffer can be lowered to 5-6
  2. Base substitution changes decreased by 60 fold and frame-shift changes decreased by 11 fold with a 3 unit pH decrease
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 D amaged template DNA Suggestions:
  1. Minimize cycle number, since repeated heating/cooling at high pH can damage template (Most templates require 25-35 cycles)
  2. Check integrity of template DNA
  3. Reduce cycling denature time to 15 - 30 seconds
Back to Top
'"/>

Source:


Page: All 1 2 3

Related biology technology :

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


(Date:12/2/2016)... ... 2016 , ... PhUSE will build on the huge success ... Day Events (SDE) to organize a multiple-day US conference. The first annual US ... the pharmaceutical and life sciences industry will cover industry standards, data science, regulations ...
(Date:12/2/2016)... KING OF PRUSSIA, PA (PRWEB) , ... December 01, 2016 , ... ... clinical research is through industry-wide collaboration, standardization and a beautiful technology experience. All three ... which convened more than 100 clinical trial leaders from over 40 sponsor, CRO and ...
(Date:12/2/2016)... ... December 02, 2016 , ... The ... to collaboratively developing improved chemistry, manufacturing and control technologies for the pharmaceutical ... with robust, probe-based sampling. , Online liquid chromatography analysis is becoming ...
(Date:12/2/2016)... YORK , Dec. 1, 2016   SurePure, ... photopurification, announced today that the Company has concluded an ... the right for a 90-day period to acquire units ... value of approximately USD 3.7 million.  ... agreement with Tamarack under which Tamarack will seek regulatory ...
Breaking Biology Technology:
(Date:11/15/2016)... DUBLIN , Nov 15, 2016 Research ... - Global Forecast to 2021" report to their offering. ... ... reach USD 16.18 Billion by 2021 from USD 6.21 Billion in ... Growth of the bioinformatics market is driven by ...
(Date:6/22/2016)... 22, 2016   Acuant , the ... solutions, has partnered with RightCrowd ® ... Visitor Management, Self-Service Kiosks and Continuous Workforce ... add functional enhancements to existing physical access ... venues with an automated ID verification and ...
(Date:6/15/2016)... 2016 Transparency Market Research ... Market by Application Market - Global Industry Analysis Size Share ... the report, the  global gesture recognition market  was ... is estimated to grow at a CAGR of ... Increasing application of gesture recognition technology ...
Breaking Biology News(10 mins):