Does anyone have an explanation for observing multiple primer insertions during site-directed mutagenesis?

12 May 2015 1 9K Report

I am using the QuikChange XL kit and I have designed the primers using the tool provided by Agilent and calculated the Tm using their equation. Also, I used IDTs oligo analyzer to check for secondary structure, and adjusted appropriately to minimize. I've tried changing thermocycling parameters but still get this result on occasion but not every time with the same template/primer pair. Any suggestions on a cause?

Xu Yang

I don't know how it happened. But it really formed 2-3 times insertions in mine and my friends' PCR results. If it happened, no need to test the Tm, just design the primer in different ways. It will work out this problem.

Badges
Science topic

More Andrew Reed's questions See All

Why is my platinum counter electrode getting dirty during electrodeposition?

I am conducting anodic electrodeposition of MnO2 onto a stainless steel substrate, using a platinum strip as a counter electrode. This is done galvanostatically, with the current held between 5...

27 February 2021 1,829 3 View

What is the significance of (isolated) high titers of IgG anti GD1a antibodies in an older patient with polyneuropathy?

Remote history of Campylobacter infection with reactive arthritis, and Spitz nevus (UMP). Arthropod exposure in Zika/ChikV endemic area prior to emergence of length dependent, sensory PN with...

18 February 2021 8,719 1 View

Can Qualtrics generate reports for survey respondents that show their answers to selected questions?

I am using Qualtrics for a Delphi survey. Does anyone know if Qualtrics can generate reports that I can send to respondents. More specifically, I want to send respondents a report of their...

13 January 2021 7,976 3 View

What are health and agricultural econmic impact of the herbicide 2,4-dichlorophenoxyacetic_acid (2,4-D) in your country?

Dear Research community, I am writing a review about 2,4-D. Google Scholar has identified 11000 publications with this herbicide as a keyword since 01/01/2018. There is plenty for me to read....

29 December 2020 1,145 6 View

What are the ethical considerations when conducting research with students who are paying for a private course?

I'm going to be teaching an online course to minors using game-based learning strategies. I wasn't to make sure I consider ethics considering students (likely their parents) will be paying to take...

12 December 2020 6,497 11 View

No host reference for metatranscriptomics?

One of the steps during the preprocessing of the data from metatranscriptomic analysis is to remove any host reads (host contamination) by comparing to host database. But what if there is no host...

30 November 2020 3,259 2 View

Primer specificity check with primer blast individually or together?

Hi, When checking my designed primers for specificity, what is the difference if I run BLASTn for each primer individually versus together (ie. forward primer separated by 20 "N" then reverse...

03 November 2020 1,421 4 View

To what extent do motorhomes and other large vehicles degrade road surfaces in relation to cars and other light vehicles?

I am studying the effects on the road system of a rural region of a drastic increase in traffic, particularly Motorhomes. My question is this, do motorhomes and other large vehicles have a...

22 October 2020 8,146 3 View

How to calculate pKa's using DFT for a molecule with three acidic hydrogens?

The molecule in question is a molecule with 3 non-equivalent hydrogens. Am I going to have to run the calculations in all of the transition states?

18 October 2020 1,524 3 View

Gibson Assembly Produces Wrong Product?

I have been using Gibson Assembly to insert a 7kB fragment into an 8kB linearized vector. After assembly many colonies form, but colony PCR on the transformation consistently shows a 1.5 kB...

18 September 2020 247 3 View

MD MOE RMSD

How to calculate the RMSD values for a MD simulation using MOE?

07 August 2021 0 0 View

MD MOE RMSD

How to calculate the RMSD values for a MD simulation using MOE?

07 August 2021 0 0 View

fatal error

When I tried to energy minimization my system, I got fatal error as below. Fatal error: Atomtype opls_116 not found Although I've already added this line: ; include water #include "oplsaa.ff/spc.itp" to [molecultype] directive in my topology.

16 June 2021 0 0 View

fatal error

16 June 2021 0 0 View

fatal error

16 June 2021 0 0 View

fatal error

16 June 2021 0 0 View

What kind of fluid could I use for a pitfall trap that also does not invalidate molecular testing?

Hi, I want to start testing pitfall trap to obtain ants samples, but I need to conduct molecular analysis on those insects. So, what kind of fluid can I use? Ethanol expires too early and I need...

03 March 2021 5,978 5 View

Can linear DNA be used as template for site-directed mutagenesis?

Is it possible to induce site-directed substitution mutation by quick-change method on linear dsDNA? or it has to be cloned in some vector? If yes, should it be treated with the Dpn1 enzyme...

03 March 2021 401 4 View

How to repeat an operation on an atom selection for multiple times in PyMol?

Hi, I am trying to construct a multi-layer fibril structure from a single layer in PyMol by translating the layer along the fibril axis. For now, I am able to use the Translate command in PyMol...

02 March 2021 4,569 4 View

New Journal, "Integrative Psychological and Behavioral Science" -- do you not know, and have you not seen, this done before?

Question to you and THEM, the New Journal, "Integrative Psychological and Behavioral Science" -- do you not know, and have you not seen, this done before? There appears to be a core problem for...

02 March 2021 3,024 2 View