I'm fairly new to qPCR and would love to find a good tutorial on primer design. I'm currently using Invitrogen Vector NTI software, but I've tried other tools and they all see to spit out a bunch of likely looking options.
Specifically, how do I decide on the most important design considerations? Should I be especially concerned about Tm or are primer dimers and hairpins a bigger worry? I've received lots of helpful advice from around the lab, but it seems like a mash of rules of thumb (avoid 4 A in a row).
If anyone knows of a good site, or has a list of rules written down, that would certainly help me.
Look at OLIGO software and website. The rule of thumb you mention about the 4As in a row is quite serious 4As are very weak bonds that will create a weak area of very low deltaG, these simply will not work 99% of the time for primers.
1. Go to [Only registered users see links. ] enter the name of the gene in the search box.
2. The search will take you to the ENTREZ search engine. Click on Nucleotide.
3. This will take you to a new webpage. Filter your results by clicking on mRNA.
4. Click on one of the links that has the desired gene.
5. Scroll down and click on CDS. This is will highlight a region of the gene sequence.
6. At the bottom of the website click on the FASTA link.
7. Copy the sequence given.
8. Open a new window and go to [Only registered users see links. ]
9. In the STEP 1 box type >species
10. Press enter then paste the sequence from that species.
11. Enter at least 4 species into the box then press submit.
12. The next page has asterisks that show you where all of the species have the same nucleotides.
13. Find approximately 300 nucleotides with the most asterisks.
14. Copy those nucleotides from the gene bank.
15. Open a new window and go to [Only registered users see links. ]
16. Click SciTools>PrimerQuest
17. Type the name of the gene in the name box.
18. Paste the nucleotides
19. Change the Design For status from PCR Primers with Probes to PCR Primers.
20. Click Calculate.
21. Add the set that has the largest Primer Product Size.
22. Put it on the wish list.
o 3’ end ends in a G or a C
o Have at least one A of T in the last three bases
o Produce and amplicon (a copied strand of DNA) of 150 to 300 bp in length.
o Primer must be between 18-25 bp in length
o Have a G/C ratio of 45-60%
o Have a Tm between the forward and reverse primer must be less than 1 degree apart
o Use free website by idt dna it is there oligo analyzer to:
• Check for hairpins which should have a Tm 10 degrees lower than your primer Tm.
• Self dimer: should have a delta G more positive than -9
• Hetero dimer: should have a delta G more positive than -9
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