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


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:

(Date:10/8/2015)... IRVINE, Calif. , Oct. 8, 2015 ... announced its expansion into the North American market with ... Asia and Europe ... a pioneer of Target Enrichment methodology and sample preparation ... or NGS, facilitates both hereditary and somatic genetic testing ...
(Date:10/8/2015)... 2015  The ALS Association, in partnership with Prize4Life, ... to revolutionize communication technology solutions for people living with ... ALS (amyotrophic lateral sclerosis) is a progressive neurodegenerative disease ... the spinal cord. Eventually, people with ALS lose the ... leads to total paralysis and death within two to ...
(Date:10/8/2015)... ... October 08, 2015 , ... ProMIS Neurosciences, Inc. (TSX: ... develop and commercialize intellectual property rights belonging to The University of British Columbia ... affords exclusive worldwide rights to intellectual property based on use of the ProMIS™ ...
(Date:10/7/2015)... ... October 07, 2015 , ... The Riordan-McKenna Institute ( ... educational seminar on the latest advances in adult stem cell therapy for orthopedic ... pm at the Hilton Dallas/Southlake Town Square in Southlake, Texas. , RMI offers ...
Breaking Biology Technology:
... Cord Blood America, Inc. (OTC Bulletin Board: CBAI), ... ) focused on bringing the life saving ... said today that Matthew Schissler, founder and CEO, in ... Company is very close to becoming cash flow positive.The ...
... CRY ), a biomaterials, medical device and tissue processing company, ... results will be released on Thursday, February 19, 2009. ... at 10:00 a.m. Eastern Time, February 19, 2009, to discuss ... by Steven G. Anderson, president and CEO of CryoLife, Inc.To ...
... INCHEON, South Korea, Feb. 5 The Centers for ... Korean,biopharmaceutical company, have signed a Letter of Intent to ... protect people exposed to,the rabies virus. , ... rabies is a two-step procedure.,First, rabies immune globulin (RIG) ...
Cached Biology Technology:
(Date:9/30/2015)... , Sept. 30, 2015  The global glucose monitoring device ... 2015. So says a new report on the industry from ... segment dominate the market, followed by continuous glucose monitoring and ... on the market for these products in its latest report, ... , ...
(Date:9/29/2015)... 2015  iDAvatars is excited to be named one of ... market. The official announcement was recently made at an invite-only ... San Francisco , where iDAvatars presented a ... Watson. "It is both an honor and a ... bring to market the cognitive power of IBM Watson in ...
(Date:9/28/2015)... 2015  The monitoring of vital signs, such ... is an essential component of patient assessment. Changes ... in a patient,s condition. However, in general care ... taken during routine observation rounds only once every ... between these observation rounds, the warning signs can ...
Breaking Biology News(10 mins):
... it can damage blood vessels and increase the risk of ... he can thin the human blood by subjecting it to ... physics at Temple University, has pioneered the use of electric ... engines and pipelines. Now, he is using the same magnetic ...
... coastal areas in Africa can rarely utilise their entire fishing ... near the coast. Research carried out by economist Kofi Vondolia ... of fish stocks is significant to fish management. ... allows foreign fishing fleets to utilise the zones further out ...
... the Virginia Institute of Marine Science (VIMS) shows that jellyfish ... altering marine food webs by shunting food energy from fish ... frequency of jellyfish blooms in coastal and estuarine waters around ... impact on marine food webs is likely to increase into ...
Cached Biology News:
... preparation of the catalytic subunit of bovine ... isolated from the yeast Pichia pastoris to ... It recognizes the sequence Asp-Asp-Asp-Asp-Lys and cleaves ... The enzyme can be used to cleave ...
... bioassays for interferons are cytopathic effect inhibition assays, ... Sample types include; tissue culture supernatents; serum from ... assays, one unit is defined as the quantity ... cytopathic effect of viral infection by 50%. ...
Biology Products: