PCR Primer Design

PCR Primer Design

  • Mahamoud Sama Cherif added an answer:
    Can someone help me with a big problem in extension PCR?

    I want to fuse a fragment with 1722 length to a 1778 fragment with very stable secondary structures.
    Overlaping primers have 53 bp.
    End primers have cut sites.
    Firstly, I have amplified two fragments and obtained specific band. I extracted two fragmets from agarose gel.
    Then I diluted two fragments to1/20. At the next step, I did SOEing PCR ..
    I have problem overlap extension pcr for two fragments from viral DNA. ....the length fragments are 1722-1778 respectively. i was amplify this fragments by taq polymerase. but I can't bind their. i used from Hot Star Taq but I can't amplify their. I would be very thankful if you help me to solve this problem.
    This situations didn't answer
    94 degree 5 minute without primers
    Put in room temperature 15-10 minute or ice
    Extension 72 degree 3 minute
    94 degree 1 minute
    60 degree 40 second 25-15-30 different cycles
    72 degree 3 minute
    72 degree 10 minute

    I would be very thankful if you help me to solve this problem.

    Mahamoud Sama Cherif · Nagasaki University- Institute of Tropical Medicine

    I agree with Imber and try some of these parameters

    1. The hold time for the extension step depends mainly on the length of the DNA template. As a rule of thumb we use 60 sec per kb.

    2. GC content of DNA template. PCR with GC-rich templates(>60%) are especially difficult.

    3. Number of cycles. The number of cycles necessary to obtain a suffient amount of PCR product depends strongly on the concentration of the DNA template. In a typical PCR, the maximum amout of product is approx. 1012 copies of the template. Starting from one copy, the most efficient PCR would reach this level in 40 cycles. Depending on the nature of the DNA template, we start with many more copies and as a rule of thumb we carry out PCR with 25 cycles for plasmid DNA and 30-35 cycles for genomic DNA.

    4. DNA template concentration. The concentration of DNA template depends on the source. Normally used concentration are 100-250 ng for mammalian genomic DNA and 20 ng for linearized plasmid DNA (circular plasmid DNA is slightly less efficiently amplified) per 50-μl reaction.

    5. Q-Solution, a novel additive that enables efficient amplification of "difficult" (e.g., GC rich) templates.


  • Mahbobeh zamani babgohari added an answer:
    Could you please help me with my PCR?

    Dear friends, 

    I am doing molecular screening for transgenic plants. I am not getting the expected band in my plants that selected on selectable medium. I'm getting a very strong band in my plasmid (as my postive control). My plants were transformed by binary vector (pBI1400) through Agrobacterium tumefaciens. The tm values of my primers are 62 and 63. I have tried with various annealing temperatures ranging from 57-63, but no result. I checked the amplification using non-transformed plant DNA with the same primers. I want to have a distinct and strong band to validate the presence of the transgene in my plants, I'm working on. Could you please help me?

    Mahbobeh zamani babgohari · Dalhousie University

     since PCR is so sensitive, you must get it if the plants are transgenic.  You can decrease annealing temperature below 57 to see what the pcr product is. Doesn't matter what primer tm is. Try different PCR reaction materials, work with Mg concentration, different Taq polymerase like Takara. Even change the thermo cycler. 

  • Rushiraj Manchiganti added an answer:
    Has anybody worked with (KAPA SybrGreen 2X qPCR Master mix), is it worth going with KAPA Sybr green?

    At present I am working with ABI, at our lab we have ABI Stepone platform!

    Rushiraj Manchiganti · Genotypic Technology Private Limited

    KAPA sybergreen as a product is pretty good.  Be careful to use the right amount of ROX.  Have a look at this link "https://www.google.co.in/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0CB8QFjAA&url=https%3A%2F%2Fwww.kapabiosystems.com%2Fdocument%2Fkapa-sybr-fast-abi-prism-tds%2F%3Fdl%3D1&ei=LT3KVPfeKcWqggThm4CwAg&usg=AFQjCNHabP0IuMWsSrP3wd27PS9Ze1q_bg&bvm=bv.84607526,d.eXY"

  • Suneelshekar Yapara added an answer:
    Can someone recommend a good resource of first strand cDNA for cloning gene by PCR?

    Dear all, I am going to clone a set of genes which express in many common tissues, eg. brain.  There are a number of companies who sell such cDNA mix. It will be helpful if you can share your expertise or good experience by using such first strand cDNA from a certain resource, to clone genes, especially genes longer than 8 kb.  Thank you.

    Suneelshekar Yapara · Gennova Biopharmaceuticals Ltd

    Try SuperScript III First Strand Synthesis System from LifeTech.

    It can handle 100bp to >12kb lengths. 


  • Raza Ali added an answer:
    Do the amplification products in MSP need to be different in size (Meth and U) in order to differentiate them within the same line of the PCR?

    Do I have to put both pair of primers (methylated and unmethylated) in the same reaction?

    Raza Ali · University of Minnesota Twin Cities

    It would be better if they would have different sizes as it would be easy to identify the semi methylated products.

  • Soumya Sankar Rath added an answer:
    Can anybody suggest me about the PCR condition for Internal transcribed spacer (ITS) region primers?

    I am working with Internal transcribed spacer (ITS) region primers for identification of some fungus. But  I am getting some problems during amplification. So if anybody can suggest me in this regard, it will be very helpful for me.

    Soumya Sankar Rath · Central Institute of Freshwater Aquaculture

    Thank you.

    I will definitely follow.

  • Elham Mahmoudian added an answer:
    What is the best tool to design primers for Real-time PCR online?

    I was used to design primers from the genscript site. Does anyone have a better suggestion for primer design for the Real-time PCR ?


    Elham Mahmoudian · Azarbaijan Shahid Madani University

    Thank you so much dear friends for your answer and helpful guides.

  • Adil Hussain added an answer:
    Is there any easier method for detecting genes in a DNA sample other than PCR ?

    When have a DNA sample and we need to detect a specific DNA sequences within the DNA extract but with faster & easier way than PCR followed by GE as a type of screening

    N.B: We haven't appropriate measures for using radio-labeled probes

    Adil Hussain · International Islamic University, Islamabad

    Sir Dr. Krzysztof Treder. Does LAMP require any special reagents or necessary components?

  • Elham Mahmoudian added an answer:
    Is it possible to limit the result of BLAST to transcripts of the special tissue?

    "bioinformatics" , "real-time PCR" , "primer design"

    Elham Mahmoudian · Azarbaijan Shahid Madani University

    as the Dhirendra Kumar said this method is useful. I had experience in it and it works.

  • Yuan-Yeu Yau added an answer:
    Does the PCR product amplified using pfu polymerase have 5' phosphate groups or do we need to treat the amplified product with PNK?

    I am trying to ligate two blunt end PCR products.

    Yuan-Yeu Yau · Northeastern State University

    1. Unlike Taq DNA polymerase, PCR from Pfu DNA polymerase will not add a nontemplate-dependent 3'-A overhangs to its PCR products. Therefore, it is not suitable for T/A cloning strategy. But, this is nothing to do with whether the PCR product has a 5'-phosphate added or not. These two should not be confused.

    2. As mentioned above by other members, primers can be ordered with the request of adding 5'-phosphate group. So, you will have the 5'-phosphate at the ends of the amplified PCR products no matter you use Taq or Pfu DNA polymerases.

    3. For blunt-end ligation, one way to minimize self-ligation is that the blunt-ended vector should be de-phosphorylated, while the insert (in this case, PCR product) has the 5'-phosphate group.

    4. In blunt-end ligations, the association of 5’ phosphate groups and 3’ hydroxyl groups is more transient than in cohesive-end ligations. Because they lack the hydrogen bond stabilization of cohesive ends. Allow for more time for ligation reaction.

  • Carmen Leitch added an answer:
    Does anyone know how to design efficient primers for real time PCR sybr green?

    I work real time PCR and I've designed a number of primers for my work, but two of them in any way I can set. I have decided to re-design their primers.

    Carmen Leitch · University of Maryland, Baltimore

    If your qPCR isn't working, aside from the suggestions about checking annealing temperature of the primers (start a few degrees below the melting temperature of the primers) you should also make sure your gene of interest is expressed in the tissue you are using and at whatever age your sample might be. 

    I also use IDT primerquest. Not only do I BLAST the primers, I also make sure they are spanning an intron/exon boundary by checking where they are on BLAT.

  • Henry Rockham added an answer:
    How can I design qualified qPCR primers?

    I was using Oligoanalyzer3.1 to check the Hetero-Dimer, all the primer pairs produced by Primer3plus have some Hetero binding locus, sometimes 3 bases, sometimes 4 bases, and there is a "maximum delta G" index showing along with the hetero binding site. I'm just wondering what is the optimal index for "maximum delta G" and should the hetero bindings be strictly avoided? Is 2 or 3 bases binding acceptable? 

    Henry Rockham · Fudan University

    Thanks a lot Maulik, i found it very helpful !

  • Shadab Kazmi added an answer:
    Is there any software for designing PCR primer reducing false priming?

    I would like to ask on a good software for designing PCR primer, offers some control to select any position on the template sequence.

    Shadab Kazmi · Aligarh Muslim University

    I agree with Biris, primer 3 is a good choice for designing PCR primers.

  • Pankaj K. Bagul added an answer:
    How to optimize qRT PCR?

    I am having difficulty optimizing the right conditions for my rtpcr. I have successfully optimized the internal house keeping gene for my pcr and it is stably expressed across all samples, with a ct value of about 18.However, whenever I do pcr for the TLR genes, only few of the TLRs seem to be working. I confirmed this by running the product on agarose gel which gave me clear bands of the expected size. Something is still not right as I don't get a good dissocation curve even for those samples which gave me product in gel. What else could be done?

    Pankaj K. Bagul · Translational Health Science and Technology Institute


    Some of the TLRs expression is really low in some cells which leads to late amplification. You can try using more cDNA sample or fresh working stock as suggested by Maulik. I was having problem of non specificity during amplification in some TLR.

    Let me know if your problem gets solved. 

  • Norman Hafner added an answer:
    Reverse transcription for qPCR 3' bias and long transcript?

    Hi all,

    I am trying to do a SYBR green based qPCR experiment on a mouse gene which has a 4kb transcript length. However, I am stuck at the Reverse transcription step because when I try to test my qPCR primers on this cDNA with a regular PCR run on gel (just to see grossly if my primers work and if I have cDNA), I cannot detect a strong single band at my predicted amplicon size (I hav tried 5 different pairs of PCR primers with products ranging from ~70-122bp) even with relatively lenient conditions. My positive control GAPDH qPCR primers DO work on my +RT template and not on my -RT template, so I know my RT reaction worked to some extent.

    I also know that my gene of interest (GOI) is expressed in the tissue I'm looking at (neural tissue) from published transcriptome data.

    I am wondering if 1) my gene of interest primers just stink or 2) my RT kit has 3' bias or 3) my starting RNA template is just low copy and therefore poorly efficiency

    Here are the exact steps of what I have done:

    1) RNA extraction using Ambion RNaqueous RNA extraction of homogenized mouse neural tissue

    2) Verified on Agilent bioanalyzer the presence of intact total RNA (~2000pg/ul, RNA integrity number of 7)

    3) Used NEB (new england biosciences) kit E3600 First strand CDNA synthesis kit (now discontinued) using "Random hexamer mix" (which is actually a mix of random hexamers and oligodT, there was no purely random hexamer reagent in the kit). Used 5-8ng of starting RNA

    4) Run a regular PCR using Taq --> disappointment :/

    Last thoughts: given the possiblity of 3' bias, I redesigned my primers to try both the 5' and 3' ends of the transcript which did not work. 

    Any advice or experience with this kit would be tremendously appreciated!!

    Norman Hafner · Universitätsklinikum Jena

    Hi Anthony,

    good news ;-) so it seems that it was indeed a concentration problem or a lower efficiency of the NEB cDNA kit.

    Good luck with the qPCR

  • Melvin Prasad added an answer:
    How can I solve this RACE PCR problem to make the band visible?

    I have problem with RACE PCR. I cannot see band. I have already changed all factors several times, but it has not helped.

    The first step (with long primer) is OK.The second step (with short primer) does not work.

    I use taq polymerase to run PCR.

    In your opinion, what should I do?

    I will be so grateful for all your help. 

    Melvin Prasad · University of Mysore

    RACE is a very tricky experiment .I use Invitrogen RACE kit, it works well. preparation of cDNA and primer specificity are very important. Keep doing with different PCR programs. I also suggest you to go for Inverse PCR to amplify unknown part of your gene.Good luck

  • Jobin John Jacob added an answer:
    How can I design degenerate primers for an unknown sequence of a gene?

    I need to design degenerate primers for the Mn SOD gene of a plant in which this sequence is not known. Can anyone please help me out?

    Jobin John Jacob · VIT University

    Hi Yordan

    i ve been following your discussion for a while. I have problem in designing degenerate primers too. Could you please explain how you design your primer? I have tried a number of software but didn't work for me. I have attached my multiple sequence alignment file here

    Kind regards


  • Sireesh Kumar added an answer:
    How to design primers for semi-quantitative PCR for the gene Prkag2 (Mus musculus)? Duplicate sequence leading to primers detecting 2 sites.
    Added to this, Prkag2 has 3 splice variants.
    Sireesh Kumar · University of Hyderabad

    thank you for answering my question Mr. Agarwal but the problem is the most of the exon part of this gene is an untranslated region and the variants of this gene is repeated three times. there fore at this situation how to design proper and specific primers.

  • Bahar Zare added an answer:
    What would be a tolerable dG value in primer design?

    dG values in primer design are very important in order to prevent homodimerisation primer dimers etc.

    A critical step in primer design is the in silico analysis of your primer pairs and amplicon.

    A number of programs can therefore be used (eg mFOLD). On several fora there is some debate about the acceptable dG values primers may have in order to be acceptable.

    Is there anybody who knows or did real wetlab studies in order to determine the accepatable cutoff  of theoretically found dGs?

    Some say that dG = -10 kcal/mol is still acceptable

    Others say a dG of =-2 kcal/mol is still acceptable

    Thanks in advance for any answer.

    Bahar Zare · National Institute of Genetic Engineering and Biotechnology


    -4 to -5 with 4 to 5 bound between two strands.

    God speed you

  • Bindu Madhavi Gopireddy added an answer:
    Are there any specific primers for the molecular variability study of Rhizoctonia solani 1-1 AG?

    I need  information regarding primers for the molecular variability study of Rhizoctonia solani 1-1 AG?

    Bindu Madhavi Gopireddy · Acharya N G Ranga Agricultural University

    Deepak Pakalapati@ thank u very much sir.

  • Subramaniyam Ravichandran added an answer:
    How can a difference in GC content in PCR primers affect PCR?

    I have a fwd primer with 37% GC content and the rev primer with 61% GC content. The Tm of the fwd primer is 55.6 C and that of the rev is 58.2 C. Upon performing gradient PCR, I saw bands at 50C and 53C but not at 52 and 54C. When I performed PCR at 50C, I did not get any bands. I am considering using DMSO to lower the Tm of the second primer, but am not sure if this could be a reason I am not getting any bands. What problems may arise from differences in GC content between primers, and could this be a reason I am getting sporadic results?

    Subramaniyam Ravichandran · Sungkyunkwan University

    Thank you all for your suggestions. 

    1. Redesigning the primer was not suitable since I was working on amplifying a GC-rich region.

    2. I repeated the experiment and got a faint band at the desired position when I performed PCR at 50C. I excised this band and performed gel elution, then used this as the template and performed PCR again using the same primers and at the same temperature, and was able to get good bands from then on. I sequenced the PCR product and found that it matches my target.

    I suspect that my problem arose because of nonspecific binding in the genomic DNA. As indicated by John Schloendorn, GC-rich primers often mispair with other GC-rich regions. When I used the PCR product as the template, the pairing region on the template was narrowed down and the primers bound to the template with more specificity.

    Once again, thank you for all your very informative suggestions.

  • Naghmeh Nejat added an answer:
    Does anyone have tips and techniques for the development of PCR Primers for highly conserved regions with Single Nucleotide Polymorphism variation?

    I'm looking for optimised methods and techniques for dealing with areas of low variation. 

    Naghmeh Nejat · Putra University, Malaysia

    Dear Asif, 

    You could do real-time PCR using High Resolution Melt (HRM) analysis technique. You would find SNP using HRM. 

  • Kanwal Naqvi added an answer:
    Can PCR be done with a 14 mer primer and a single strand template of DNA nearly 100 bases long ?

    Dear all

    I want to ask i have only one primer and only one DNA template. I want to extend the primer through PCR. DNA template is also single stranded. Is PCR possible , without any complications, as in all literatures people use two primers and a double strand dna template. I dont want multiple products so i want to amplify one single stranded DNA.

    Kanwal Naqvi · Quaid-i-Azam University

    Dear Vandana

    i suggest to use ds DNA, it will be convenient for you otherwise you will have to look after many points. Regarding primers 14bp is very short and will result in non specific amplification. it should be minimum 18bp long and 22 is good length.

  • Brendan Delange added an answer:
    How can I increase the specificity of long-tail PCR primers?

    I'm using tailed primers: 20-nt of priming sequence followed by a 10-nt tail. I know to use the annealing temperature (T_A) of the priming sequence (not the full 30-nt sequence) when designing my PCR protocol, but that should only be relevant for the first few rounds of PCR. After that, the tail will have been incorporated into the template, and using the lower, priming-sequence-only T_A will allow for more spurious side products. I'm thinking that after 10 rounds of PCR, I will increase the T_A to that of the full-length, tailed template, to increase the specificity of the reaction. So 10 round at T_A1, 20 rounds at T_A2. Is this solid thinking, or is there a consideration that I am missing?

    Brendan Delange · University of Michigan

    Thanks everyone! I'm going to start with the estimated T_A (for Phusion, modified Breslauer thermodynamics) and use a touchdown protocol if I have difficulty with non-specific products.

  • Ravi Mehndiratta added an answer:
    Can anyone help in desigining primers for AGPase gene large subunit in wheat for real time pcr analysis ?

    Can anyone help in desigining primers for AGPase gene large subunit in wheat for real time pcr analysis ?

    Ravi Mehndiratta · CCS Haryana Agricultural University

    Sir I wanted to know the basics as from the sequence how to go about it .... that will ber very thankful of u

  • Mitzi Kramer added an answer:
    How to design primer which will pick up different splice variants with Primerdesign for different transcript variants?

    Dear members,

    you all have helped me along very much so far. I have now encountered yet another challenge: I need to design primers for 15 proteins which cover more than one splice-variant of the gene in question (ideally all splice variants).

    As i know so far, Ncbi_Nucleotide lists all known variants and i can pick a primer via the button "pick pimers" for each specific primer. However, findig a primer wich picks up a lot of different splice variants may be just a question of luck and a lot of trial and error in my eyes.

    Is there any programm/website or loophole which could help me along?

    Again, I will deeply appreciate your answers.

    Greetings from Munich


    Ps: Because of this community I could actually get my project going last week. To all members: THANK YOU SOOO MUCH!

    Mitzi Kramer · Ludwig-Maximilians-University of Munich

    Thank you so much for all you answers!!

    They helped me alon a lot!!!

    lots of love


  • Elham Mahmoudian added an answer:
    Primer and probe design for gene overexpression?

    I want to check expression level of a gene of interest in an organism. I am new to real time-PCR. Could one kindly guide me how to proceed. I already have designed primers for amplification of the gene of interest. Do I need to additionally design any probes or SYBR itself will dol? A step by step explanation and guidance would be helpful.

    Elham Mahmoudian · Azarbaijan Shahid Madani University


    as Yunfu Lin  said you need just primer. His guidance is excellent . your design is important.  your primers' Tm must be very near together. the size of your primer is important too.If this is your first time,it is better to have a guide that stand by your side.

  • Yue Keong choon added an answer:
    Which one is the best tempIate for qPCR primer standarization?

    I have tried aII combinations of primers and Temp. for genomic DNA standarts, but I havent got a good efficency, even tho the primers have been reported having a very good efficency using genomic DNA.  I have the information that my DNA couId be fragmented, but Im wondering that maybe this time I wiII extract RNA instead of DNA , to do a standarization with cDNA. Any recomendations or thoughts about it? which one of the tempIates have gave you better resuIts?

    Yue Keong choon · University of Science Malaysia

    i use cDNA to run qPCR. First, you need to check the RNA integrity by RNA BioAnalyzer. Design your primer by using GeneScript by adjusting certain parameter such as short basepair (75-150bp), GC content, etc. Test your primer by running in conventinal pcr whether cDNA does amplify your primer before go for qPCR. Then, you can construct standard curve for each of your primer in order to check primer efficiency. 

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