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Primer design
Primer design
Primer design
Primer design
Primer design
Primer design
Primer design
Primer design
Primer design
Primer design
Primer design
Primer design
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Primer design

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how to design primers

how to design primers

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  • 1. How to Design PrimersFor PCR using Primer3 website
    Yousef Alhashem
    2011
  • 2. PCR
    Polymerase Chain Reaction
    in vitro amplification of DNA using heat stable DNA polymerase enzyme.
    Three cycling steps:
    Denaturation
    Annealing
    Elongation
    At the end of each cycle the amount of DNA is doubled.
    Reagents needed:
    Template (DNA)
    Polymerase (e.g. Taq polymerase)
    Primers
    Nucleotides (A,T,C,G)
    Magnesium, potassium, and buffer
    DNA
    Denaturation
    Annealing
    Elongation
  • 3. Primers
    Short sequence of nucleotides
    ~20 nucleotides
    Required to initiate DNA synthesis by polymerase.
    Good primers:
    About 20 nucleotides long
    50-60% are G+C
    Have melting temperature ~60
    No more than 3 G or C at the 3’ end
    Not self complementary
    DNA
    Denaturation
    Annealing
    Elongation
  • 4. Primers Design
    Use Primer3 website to design primers for known regions of DNA
    http://frodo.wi.mit.edu/primer3/
    Primer3 developed by Steve Rozen and Helen Skaletsky.
    Primer3-web is maintained by Steve Rozen.
  • 5. Obtain DNA
    Find the DNA sequence of the region you want to amplify
    You can use the NCBI website for that.
    http://www.ncbi.nlm.nih.gov/
    Select Gene database and search for you gene of interest
  • 6. Obtain DNA
    If you search for klf1 for example, you will get klf1 genes in several species
    Select the correct species. I select mouse (Musmusculus) klf1.
  • 7. Obtain DNA
    If you search for klf1 for example, you will get klf1 genes in several species
    Select the correct species. I select mouse (Musmusculus) klf1.
  • 8. Obtain DNA
    Go down the page until you reach genomic section
    Select GeneBank if you want to learn more about the structure of your gene.
    Select FASTA to get only DNA sequence of the gene
    Go down
  • 9. Obtain DNA
    Copy the region of DNA you want to amplify.
    Go to Primer3 website, http://frodo.wi.mit.edu/primer3/
  • 10. Primer3
    Paste the DNA sequence into the provided space
    Tell the website where do you want you primers to be by adding “[“ and “]” around the sequence of interst.
    Tell the website what is the size of amplicon you would like to see.
    Hit “Pick Primers”
  • 11. Primer3
    You will get the best pair of primers at the top of Primer3 Output screen.
    The primers’ sequences , the melting temperature as well as other information will be shown.
    You need to make sure your primers are specific using NCBI blast engine.
    http://www.ncbi.nlm.nih.gov/tools/primer-blast/
  • 12. T.h.a.n.k
    Y.o.u

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