Query regarding Primer design

DNA Sequencing
1

Hi

I am struggling to design my primers for TAF15-201.I am supposed to do it for 2 consecutives exons, but I don’t know what I am supposed to do with introns Do I need the full intronic sequence? how do I link both exonic sequences?

Then once in my primer design tool, and knowing that I need to amplify at least 30bp upstream and downstream of the exons encoding TAF15-201 What are the ranges for my forward and reverse primer what is my pcr size ?

where do my primers sit in my sequence ?

2

Hi Daniel You can follow these steps for achieving results:

Step 1: Retrieve Sequence Data

  • Obtain the full sequence of TAF15-201 including exonic and intronic sequences from a database like NCBI or Ensembl.
  • Identify the exon-exon junctions of the two consecutive exons you want to amplify.

Step 2: Define the Target Region

  • Determine the length of each exon and the adjacent intronic regions.
  • Set your target amplicon to include:
    • 30 bp upstream of the first exon.
    • 30 bp downstream of the second exon.
    • This means your amplicon will cover both exons and parts of the intronic regions.

Step 3: Primer Location

  • Forward Primer: Place this primer 30 bp upstream of the start of the first exon. It should be within the upstream intron or the 5’ untranslated region if available.
  • Reverse Primer: Place this primer 30 bp downstream of the end of the second exon, within the downstream intron.

Step 4: Calculate PCR Product Size

  • Measure the total length from your forward primer binding site to the reverse primer binding site. This includes:
    • The length of both exons.
    • The intronic region between the two exons.
    • The 30 bp flanking sequences.
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