PCR Primer Design

PCR Primer Design

  • Sabine Strehl added an answer:
    Can anyone suggest how I can create a positive and negative control in PCR reaction?

    I have a DNA sample and I don't know is it good or bad, I will do PCR for it.

    I want to be sure that the bands that will appear after PCR are the wanted amplicon and not a primer-dimer. I need a positive and negative control in my PCR but I don't know how to make these controls in PCR.

    Any help would be gratefully received. 

    Sabine Strehl · Children's Cancer Research Institute

    I would suggest that you seek advice from your supervisor!

  • Noha Said added an answer:
    What is the difference between PCR-tetra primer ARMS, PCR-AS, and PCR-CTPP?

    I want to know the difference between PCR-tetra primer ARMS, PCR-AS, and PCR-CTPP. I want to use the most simple one in the genotyping.

    Noha Said · Zagazig University


  • Daniela Iancu added an answer:
    Can you confirm the results of my MPLA experiment by Long PCR?

    Hi everybody! I am carrying out an MLPA experiment and I have to confirm my data.

    I have chosen Long-PCR approach to confirm the presence of deletions and to define their limits, but I have some doubts: can I achieve my aim if the PCR primers are designed upstream and downstream of the first probe and of the last probe related to the deleted region, respectively?

    For example, the deletion encompass the exon 2, 3 and 4: the primer 2f is designed upstream of the probe number 2 and the primer 4r downstream of the probe number 4. So, using this conditions am I able to amplify and sequence the correct region to analyse the deleted region or I have to use the primer 1f and 5r?

    I hope it's clear...I am looking forward hearing from you!

    Daniela Iancu · University College London

    Yes, it is true. However, the probes are located somewhere in the middle of the exon (or close to it) so theoretically the deletion could be in the same exon but in 5' (or 3' respectively) from the recognition site of the probes. Most probably the breaking point is located in the intron and this is why it is more likely to have a better result for the primers containing the first non-deleted exon on each side. Several pairs of primers to cover the entire region might be necessary.

    Additional splicing modifications can introduce some intronic sequences in the mRNA and this you will not be able to see at the genomic level. If you detect the breaking point you can use the sequence to predict the potential splicing effects but the proof in this case stays in the cDNA sequencing.

  • Chenhui Wang added an answer:
    Why do I get two bands with RNA probes using PCR products?

    I made RNA probes with primers including T7 and SP6, after I got the PCR products, the sequencing came back to confirm that it was what I want. then I purified the products and ran the gel again to check the band and did reverse transcription, but when I run a gel to check my probe, I got two bands, one was what I want ,the other one was shorter, I don't know what happened?

    Chenhui Wang · National Institute of Biological Sciences, China

    I would suggest that you check whether the linearized template has a 3'-overhang.  It has been reported that 3'-overhangs generate spurious transcripts and should been avoided. Therefore, it is preferable to use restriction enzymes which generate 5-overhangs or blunt ends. I hope that this is going to help you.

  • Julia Powers added an answer:
    Does anyone know of a published (proven) primer set for human mitochondrial 16S ribosomal RNA?

    I am trying to find a mitochondrial DNA 'housekeeping gene'.

    Julia Powers · IT Sligo

    Thanks Antonio, but my institution doesn't have access to Wiley.  Could you copy the actual primer sequences and message me if you have time? The fact that nuclear sequences are excluded is a bonus.

  • Khawar Sohail Siddiqui added an answer:
    What are the available, easy-to-use primer designing tools or programs?
    I wonder if anyone can provide me with sources of freely available primer designing tools or softwares. I have trained on using Primer3 tool available at GenBank, but I noted that there are many other tools that make one confused about the best alternative. I would be very grateful if you can provide me with any tools and sources related to simple primer design.

    Thank you in advance!
    Khawar Sohail Siddiqui · King Fahd University of Petroleum and Minerals, Dhahran, Saudi Arabia

    which one is the easiest for novice? I want to design guessmers for bacterial lipases which have not been sequenced before. 

  • Artur Burzynski added an answer:
    How can one trim primer sequences?


    is there any software to trim off primer sequence from sequence (full length sequence). Is it possible to do so using BioEdit? or is there any software to find/place primer sequence and trim them. 

    I need to trim primer sequence from full length sequences where I used forward and reverse primers. I can keep the last 3 nucleotides from each forward and reverse primers. Is that also correct method to keep 3 or trim all of the primer sequence? 


  • Younes BOUALLEGUI added an answer:
    Is it possible to make RT PCR primers directly from DNA sequence to estimate the expression level?

    I transformed Hepatitis B surface antigen genes for edible vaccine development in tomato. I want to estimate the expression level of that antigen through RT PCR. Someone told me I may use the sequence of my transformed gene directly and design a primer which results in product size under 200 bp instead of going for mRNA sequence, and then making complementary DNA . Can someone guide me as to how should I do it?

    Younes BOUALLEGUI · University of Carthage

    I can conclude, you have to deseign your primers from the transformed gene, you have to use the cDNA as template to pick up your primers and you must be sure that your primers picked from the good area ( not non sequenced genes=introns) 

  • Heba Ebeed added an answer:
    How does one detect which primer among the primer pair is contaminated?

    I am getting a proper PCR product band in agarose gel on my negative or non template sample. So it is confirmed that my primer is contaminated.

    But, I want to know that which one is contaminated? Is this my forward or reverse primer, or both of them are contaminated. Is there any way to know the specific primer for this disturbance ?

    Heba Ebeed · Faculty of Science, Damietta University

    another assumption may water is also contaminated try to use another nuclease free water

  • Joachim Mergeay added an answer:
    Does anybody has experience using SP Designer (Villard & Malausa, 2013) for designing species specific primers?

    I am considering to design species specific primers to identify species from fecal samples. Would you like to suggest me which software is the best ?

    Joachim Mergeay · Research Institute for Nature and Forest

    you may want to use ecoprimers http://www.grenoble.prabi.fr/trac/ecoPrimers

    Check out the metabarcoding.org page for more info. 


  • Ivan Brukner added an answer:
    16S qPCR primer efficiency?
    I want to get the relative abundances of different members of a bacterial community (gut sample).

    Unfortunately, the efficiency of my reactions (determined by 10x template dilutions) is only around 80%. Does anybody have experience with these problems? The choice of specific primer binding sites is, as you can imagine, very limited (I have 3 similar Lactobacilli in my samples).
    Ivan Brukner · Jewish General Hospital

    this is not only DNA purity problem, but non-universality of universal primers

    diluting complex sample one changes  distribution of different species, leading toward non-linear Ct increase

  • Akhil Raj P added an answer:
    Does anyone have experience with PCR primer designing?

    I have to amplify a 1568 bp dna by PCR. when designing primers using primer3. I am unable to obtain primer for a larger sequence. It shows primer sets which amplify small regions. However I need to amplify the entire 1568bp region. please help

    Akhil Raj P · Rajiv Gandhi Centre for Biotechnology

    Try any other software like Primer Premier, NCBI Primer Blast..

  • Karin Glaser added an answer:
    How do I design species specific primers?

    I would like to develop specific primers for detection of basidiomycetes from soil. Any suggestions on how to do this?

    Karin Glaser · University of Rostock

    Specific primer design is a big issue.

    First step should always be: searching literature.
    Than you think about which DNA-region to choose (like Jincai said). For Fungi the ITS-region is often choosen, because they are difficult to differentiate on basis of the 18S.

    in addition to Jincai I want to mention that their are programs that help you. For example ARB can also recommend specific primers.

    After you find potential good primers you can do a quality check: for homo- and heterodimers, GC-clamps and similar annealing temperature for both primers. Also you can find some rules in the internet, like not having to much repeats in the primersequence and go for GC-rich 5'-ends.

    The verification of the specificity is the next step. Check not only for false positives but also for false negatives.
    If one primer binds at a non-target organism it isn't that bad if the second primer does not bind.

    If the primers than pass the culture test (5. at Jincais list) you would try them on real samples and play a lot with PCR conditions. And than start cloning and sequencing. After this step you can be sure that you've developed specific primers.
    This is worth a publication :)

    Hope I could help a bit, Karin

  • Shin Wei Tie added an answer:
    Anyone has come over a weird melt curve and gel image from real time RT-PCR?

    I have been trying many times and still failed to give single peak in melt curve. I have designed a new primers to detect all 6 variant of my target gene. Please refer to the blast result as attached. I have done Ta optimisation but the result is weird. My desired PCR products should be 137bp but the gel image didn't match. Final concentration for both forward and reverse primers are 0.5uM. The one step real time RT-PCR is as below:

    Cycle 1: (1X)
    Step 1: 42.0 °C for 30:00.
    Cycle 2: (1X)
    Step 1: 95.0 °C for 05:00.
    Cycle 3: (40X)
    Step 1: 95.0 °C for 00:10.
    Step 2: 60.0 °C-65.0 °C for 00:30.
    Data collection and real-time analysis enabled.
    A: 65.0 B: 64.8 C: 64.2 D: 63.3 E: 62.0 F: 61.1 G: 60.5 H: 60.0
    Cycle 4: (1X)
    Step 1: 95.0 °C for 01:00.
    Cycle 5: (1X)
    Step 1: 55.0 °C for 01:00.
    Cycle 6: (61X)
    Step 1: 65.0 °C-95.0 °C for 00:10.
    Increase set point temperature after cycle 2 by 0.5 °C
    Melt curve data collection and analysis enabled.

    Shin Wei Tie · Universiti Tunku Abdul Rahman

    I have done primer optimisation. 

    Lane 1: 50bp Ladder

    Lane2: 300nM Forward, 150nM Reverse

    Lane 3: 150nM Forward, 300nM Reverse

    Lane 4: 150nM Forward, 150nM Reverse

  • Jai Ghosh added an answer:
    Does anyone have experience with RT-PCR?

    Which one is better in synthetic of first strand cDNA during reverse transcription: Revers Primer or Forward primer or both? I believe the Revers one is useful. Is it quite true? 

    With many Thanks..

    Jai Ghosh · Shivaji University, Kolhapur

    I feel that bothe the primers are very, very useful. Do not get carried away with the data of the reverse primer alone. In case of Reverse transcryptase, both the primers' data can answer many querries.

  • Shin Wei Tie added an answer:
    Can anyone help me understand 2 Peaks in a Melt Curve in RT-PCR?

    I have a dissociation curve like this as attached. May I know if another peak is primer dimer or another products?

    I blast my primers, one of the variants (total 6 variants) can only be amplified by reverse primers but not forward primers. From the agarose gel image, the primer dimer band in NTC is lower than the second band. The first band on top is the desired band. Is it possible the second band is the amplified fragment with only reverse primers as the intensity of that band seems like same with the one on top of it?

    Shin Wei Tie · Universiti Tunku Abdul Rahman

    Dear Sushma & Britta,

    Thank you very much for the advice and suggestion. Appreciate it very much. I will study and try out on new primers. All the best.

  • Bailey Massa added an answer:
    What is the reason for getting a fragment of DNA rather than a vector after mutagenesis PCR reaction?
    I have done a lot of mutagenesis and I have a protocol that usually works 90% of the time in pET21d and pGEX6P-1 vectors. I have started mutating sequences in mammalian vectors such as pcDNA3 and 5 and for one of the mutagenesis reactions I always get a band at about 2 or 300 bps. I have increased the annealing temperature, increased the elongation time, but I always get this fragment produced. When I transform the reaction into DH5alphas I don't get any transformants. I have also redesigned the primers making them slightly longer to 45 nucleotides with a Tm of 78. I am not sure what else to try.
    Bailey Massa · Newcastle University

    Turns out it was the primers- they seemed to be binding to the complementary primer strand in multiple ways. I have changed the sequence in the regions of possible pairing (made sure to not change the amino acid sequence) and it was successful!

  • Graham Williams added an answer:
    What are the best methods, including primers and probes used for TaqMan Real Time PCR for the quantification of Leishmania infantum minicircle DNA?

    Is there a TaqMan Real Time PCR method developed for the quantification of Leishmania infantum minicircle DNA in peripheral blood of naturally infected dogs

    Graham Williams · University of Huddersfield

    Try the following reference, if you haven't already.

    Charles Mary,* Franc¸oise Faraut, Laurie Lascombe, and Henri Dumon "Quantification of Leishmania infantum DNA by a Real-Time PCR Assay with High Sensitivity" JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2004, p. 5249–5255 Vol. 42, No. 11

  • Korcan Ayata added an answer:
    Does anyone have mouse real-time PCR primer sequence that are validated and specific for a cytoplasmic enriched target?

    Cytoplasmic real-time PCR primer sequence?

    Korcan Ayata · University of Freiburg

    The statement "very cytoplasmic enriched" is highly depends on the cell/tissue type as well as the state of the cell, because the amount of transcript in the cytoplasm is directly related to cell type and activity. You would find many myelin basic protein transcripts in an oligodendrocyte while an astrocyte that sits next to it, won't have any.

  • Stella Evans added an answer:
    How do I design primers that will distinguish between highly homologous transcripts?

    I'd like to perform RT-PCR to distinguish between transcripts that are about 95% homologous.  Does anyone have experience in determining how unique the primers must be in order to detect only the transcript of interest?  For instance, if the sequence to which the primer will anneal differs for about 6 out of 20 bases between the 2 homologous transcripts, is that enough?  Is there some minimum threshold in terms of the number of unique bases?

    Stella Evans · Bryn Athyn College of the New Church

    Yes, looking at the 3' end of the transcripts is a great idea.  But, in this case, the 2 transcripts are still highly homologous in this region.  Sequencing is also a great idea.  I'm hoping that I can find a unique restriction site in the amplicon to verify its identity.  Thanks again for all of the feedback!

  • Kevin Bonham added an answer:
    Best way to design Microbial species-specific primers for qPCR?

    I'm trying to design some PCR primers to quantify the abundance of known bacterial species within a larger, defined community. I have sequences for all of the bugs, but very little experience w/computational tools to analyze them. All of my experience with qPCR is looking at mammalian mRNA expression

    Wondering what's the best approach. Should I...

    a) Align their 16S region and pick unique unique sequences?

    b) Use some sort of software (if so, what?)

    c) Something else?

    And are there any special considerations I should be considering?

    Kevin Bonham · Harvard University

    Whole genome sequencing - we don't have a complete contig but pretty good coverage over the whole chromosome. 

  • Britt Merlaen added an answer:
    Any suggestions on primers design for 5' RACE PCR?


    I am designing gene specific primers for 5' RACE PCR. I am using the protocol in the attachment. I can see that the GSP-RT primer needs to be antisense regarding the mRNA sequence, but do GSP1 and GSP2 need to be sense or antisense?

    Thank you!

    Britt Merlaen · Ghent University

    Thanks a lot!

  • Oliver W Bayfield added an answer:
    Does anyone know whether is it reliable to trust an assembled contigs sequence?

    I have a sequence (assembled contig) in which bottom 3rd frame shows homology to a known protein. Is it reliable? How should I go about designing primers?

    Oliver W Bayfield · The University of York

    Hi N James
    You could confirm the contigs have been assembled correctly by sequencing across the join (with sequencing primers flanking the join region).
    Assuming you just want to give it a go and based on the presumption the contigs are assembled correctly, design forward and reverse primers that amplify your target gene (e.g. from start codon to stop codon, or from promoter to terminator etc., depending on your application).
    Then you can sequence the whole putative gene.
    Baring in mind mind you might want to amplify between regions slightly  more up and down stream of the gene if you'll be sequencing by synthesis using primers up and down stream of the target gene. Or if you're cloning into a vector, use the vectors promoter and terminator as flanking sites for sequencing. If it's a long sequence (>800bp) you might need an internal forward primer for sequencing in addition to the main forward and reverse primers, as in my experience sequencing by synthesis sees a decrease in signal after about ~600bp (varies +/- 200 depending on sample quantity and factors I'm unaware of - we send our samples for sequencing to GATC in Germany), and this unclear region can likewise have too weak a signal in the reverse sequence chromatogram also - meaning the sequence of this middle region is unclear. You may need more than one internal primer for particularly long targets (I assume this depends on the reach of the polymerase used in sequencing - perhaps someone can confirm this).


  • AHSAN SATTAR SHEIKH added an answer:
    Can anyone assist with U.V primers QRT-PCR Primers?

    Does anyone know of a decent set of real time PCR primers that can be used with cyber green for the detection of any tRNA?

    AHSAN SATTAR SHEIKH · University of Lahore

    Dear Jordan, I have used number of primer design tools. These include LUX-D invitrogen primer design, Primer 3, QuantPrime, GeneFisher etc etc. Unfortunately LUX D software has been withdrawn whish was special for LUX primers. I think for you, QuantPrime will be of great help. Some other softwares are available that also show you the secondary structure in the Primers and target sequences that can also help you to get optimized primer sets. One suggestion is to use Tm temperature, if possible, around 60 - 65 C and amplification of 100-150 nucleotides.

  • Judith Weerts added an answer:
    Are there any difference between real time PCR primers and normal PCR primers?

    gene expression analysis

    Judith Weerts · Academisch Medisch Centrum Universiteit van Amsterdam

    I have very good experience with the free online program by MIT. I design for 100-200 bp products.


    The primerblast program from NCBI uses the same interface but also blasts the sequences at the same time. On Biorad real time PCR machines I usually have great efficiency at the first go. For cDNA I always design intron spanning primers.

  • Rinaldo Batista Viana added an answer:
    If we have primers and primary antibodies for bovine species, can we use these primers and primary antibodies also for the buffalo?
    In our lab, we purchased the primers and primary antibodies for the bovine fibroblasts, but now want to conduct the experiment in buffalo fibroblasts. Can we use the same primes and antibodies?
    Rinaldo Batista Viana · Universidade Federal Rural da Amazônia (UFRA)

    I believe we must consider that agents / antibodies in diagnosing using these primers. Some agents that affect cattle do not behave the same way in buffaloes. And certain diseases with similar symptoms can separate agents, which produce antibodies will be specific for each species.

    Rinaldo Viana

    buffaloes health

  • Deanna Larson added an answer:
    Does anyone have knowledge on genotyping for APCmin mice?

    Hi All,

    Does anyone have a good protocol for genotyping APCmin mice? I have the primers from the paper below, but was wondering if anyone has recommendations for the PCR setup (i.e. annealing temp, cycle number and so on). Thanks much. 


    Deanna Larson · Utah State University

    This is what we used.... Hope it helps.  We used amplitaq Gold MM.

  • qi Xu added an answer:
    How can I add a palindrome behind my target DNA fragment by PCR? How the primer be designed to avoid the formation of dimers and hairpins during PCR?

    I have a plasmid carried mCherry. Now I want to amplify the mCherry gene sequence from this plasmid, and at the same time add a GAL4 gene sequence just behind mCherry. But, GAL4 gene sequence(17bp) is a palindrome, thus, we cannot just by adding the gene sequence of GAL4 in the reverse primer to achieve our goal. So, how can we achieve our goal by PCR or is there any other new method can help me?

    qi Xu · South University of Science and Technology of China

    Do you mean that we add the first half of the palindrome for the first time. And then, on the second PCR, we add the second half of the palindrome just behind. But, for the  second time, the primer we used becomes a palindrome again, since it must include the first half of the palindrome, and also the second half as templete for this PCR. Or you just mean other ways?

  • Nelson Carvalho Filho added an answer:
    Can anyone help with annealing troubleshooting?

    If you increase annealing temperature, let say from 60 to 63 degrees, will Ct values from the same cDNA concentrations be changed?

    It is just a matter of setting up your machine. The one we have is a CFX96 from bio-rad. http://www.bio-rad.com/pt-br/category/real-time-pcr-detection-systems. 

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