Oct 13, 2020
- 1UCSC
- UCSC BME 22L
Protocol Citation: Ikenna Anigbogu 2020. Protocol 1: PCR. protocols.io https://protocols.io/view/protocol-1-pcr-bnc6maze
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 13, 2020
Last Modified: October 13, 2020
Protocol Integer ID: 43134
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Before start
To build PCR primers, it is as simple as uploading a FASTA file to the PrimerQuest Tool website. It works either by uploading manually a sequence, FASTA entry, downloading NCBI or Accession ID, or uploading an excel file with sequences on it.
Find a Sequence
You will need to look up the gene for the sequence you are trying to amplify and download your file (any type described above)
Go to PrimerQuest Tool
You will need to go to the PrimerQuest Tool webpage, https://www.idtdna.com/Primerquest/Home/Index, in order to insert file type of choice
When the file is uploaded, it will direct you to the screen above. You can choose pre-selected primer regions that the software finds
Select a primer
- You are able to select primers based on the size of the amplicon produced or the actual size of the primers.
- To look more in depth on a primer click the link. It gives the start and stop position of the primer in respect to the file sequence, the melting temperature of the primer, and the GC content of the primer
Take a screenshot of potential primers found on IDT then upload them to your lab notebook
Confirm your primer. The confirmation page will compile all of the necessary primer information onto one page so that you can have one last check of the aspects of your primers before you order. When you are confident in your choice, save the primer information and send them out to be synthesized.