How to Design PrimersFor PCR using Primer3 website<br />Yousef Alhashem<br />2011<br />
PCR<br />Polymerase Chain Reaction<br />in vitro amplification of DNA using heat stable DNA polymerase enzyme.<br />Three ...
Primers<br />Short sequence of nucleotides <br />~20 nucleotides<br />Required to initiate DNA synthesis by polymerase.<br...
Primers Design<br />Use Primer3 website to design primers for known regions of DNA<br />http://frodo.wi.mit.edu/primer3/<b...
Obtain DNA<br />Find the DNA sequence of the region you want to amplify<br />You can use the NCBI website for that.<br />h...
Obtain DNA<br />If you search for klf1 for example, you will get klf1 genes in several species<br />Select the correct spe...
Obtain DNA<br />If you search for klf1 for example, you will get klf1 genes in several species<br />Select the correct spe...
Obtain DNA<br />Go down the page until you reach genomic section<br />Select GeneBank if you want to learn more about the ...
Obtain DNA<br />Copy the region of DNA you want to amplify.<br />Go to Primer3 website, http://frodo.wi.mit.edu/primer3/<b...
Primer3<br />Paste the DNA sequence into the provided space<br />Tell the website where do you want you primers to be by a...
Primer3<br />You will get the best pair of primers at the top of Primer3 Output screen.<br />The primers’ sequences , the ...
T.h.a.n.k<br />Y.o.u<br />
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Primer design

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Primer design

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