Last updated: 09/28/22

RT-PCR Protocol (Gabrielle):

Primer design:

  1. First check PrimerBank: https://pga.mgh.harvard.edu/primerbank/
    1. Search by: NCBI Gene Symbol
    1. Species: Human (or mouse)
    1. For text: put gene name
  2. If nothing comes up: search the literature
  3. If find nothing in literature: time to design your own
  4. After primers have been selected place them into an EXCEL primer tracking sheet with the following information
    1. Create a unique name for each primer
      1. Include: Species h or m, for humans use all capital letters, for mouse only first letter is capital (ie hGAPDH vs hGapdh)
      1. Amplicon size if known or another unique identifier like author last name
      1. F or R to identify the forward or reverse primer
    1. Input the sequences
    1. Amp size
    1. Temperature (Tm)
    1. Location
    1. Where the primer is from
    1. Ordered date
  5. After the primers have been logged, order them on IDTDNA: https://www.idtdna.com/pages
  6. Log in with Lab log in
  7. Click on the orange Quick Order button under DNA&RNA
  8. Input the unique name you created under item name
  9. Scale: 250nmole
  10. Formulation: None
  11. Purification: Standard Desalting
  12. Copy and Paste sequencing from tracking sheet
  13. Once all primers have been added click add to order
  14. Then follow buttons to place order
  15. Make a note of the order number in case of issues
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