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
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
