Designing Oligonucleotides for qPCR
From SDMRC
Summary
We design primers get an optimal PCR. It is an important step. If you use any primers without design, the amplification may not work or give you skewed results
Method
For example: The gene Rapsyn
- Go to: lifescience.roche.com Assay Design Center
- Assay design center
- Enter species name (Mus musculus)
- Specific target
- Write gene name
- Automatically select is checked (design multiple is unchecked)
- Hit design and wait
It will give you two sequences of primers (left and right) Bottom of the page_more assays. Take first primer listed (make sure gene name is in title)
- Open an excel data sheet
- 1st column_Rapsyn_qFor (forward= left and rev= right)
- 2nd column_Rapsyn_qRev (repeat for each gene For and Rev)
- Copy and paste the sequence in excel
- Check sequence matches primer
- Copy sequence
- Go to Blastn Blastn
- Paste sequence
- Choose search serch set_ mouse genomic + transcript
- Hit blast, look at the 100% identity search option (don’t use predicted) and repeat for each
- Type “validated” for each confirmed sequence
- Add the number of nucleotides (ex. 75) found in the amplicon in the excel sheet
Second Way to Design
Summary
If the life science basic assay design isn't working design primers using this protocol instead
Method
- Go to ensemble.org (gene site)
- Search “mouse”
- Type in the gene name
- Hit “go”
- Look for gene transcript (qPCR/RNA)
- click the first one listed with the gene and strain we are using
- you’ll see a table with transcripts and proteins
- Click CCDS (coding sequence) use this one
- Website will change to NCBI, double check at the top that you have the right gene
- Scroll down, nucleotide sequence, this are the exons (in black and blue)
- Copy and paste the nucleotide sequence into a word document (label document with gene name)
- **from here design the primer ~ 20 base pairs**
- PCR product is around 200 bp, for good quantification
- GC count should be around 40-60% of total nucleotides
- Google GC content primers (Primer3plus)
- Design primers to overlap exons to double check there’s no introns (take 5-10 nucleotides before)
- Blast to verify, if only one is connected with primer its okay
