This document provides instructions for designing primers for PCR using the Primer3 website. It explains that PCR involves cycling between DNA denaturation, annealing of primers, and elongation to amplify a target DNA sequence. Good primers are around 20 nucleotides long with a GC percentage of 50-60% and melting temperature around 60°C. The document guides obtaining a target DNA sequence from NCBI and using Primer3 to design primers, specifying amplicon size and target region. It recommends checking primer specificity using NCBI BLAST.

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