View source for Designing Oligonucleotides for qPCR

==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 [https://lifescience.roche.com/en_us/brands/universal-probe-library.html#assay-design-center 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 [https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch 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