Hello
I did restriction digestion and ligation of a huamn gDNA construct and I have now the pure plasmid DNA and want to check the sequence alignment.
I've read the things that should be considered before designing a primer for PCR such as GC content and melting temp ..etc
Just wondering about the protocols for designing a primer for sanger DNA sequencing ?
Thanks
Hi Abdulrahman,
For Primer Design.
in attachment, I have put a tutorial that shows the basics of primer design that goes though specificity, melting tempt, primer length, product size, secondary structure and GC clamp. It shows you how to use two widespread tools (Primer3 and Oligocalc).
In addition, I always like to check my primers with an in silico PCR (https://genome.ucsc.edu/cgi-bin/hgPcr) to ensure that I have not made an error in the sequence I will order, that the primer is specific and that it maps exactly where I want it to.
For Sanger sequencing.
I generally design primers for each 800 bp span (the signal is generally good at that length). ---> If you have a large insert, you'll need to design overlapping sequencing products, I personally like to have about 50 to 100 of overlap.
You'll want to design the primers at least 20-30 bp upstream (forward) or downstream (reverse) of your region of interest since Sanger sequencing signal is poor for the first few bases.
When I want to confirm a mutation (or absence), I always sequence the product with a forward and reverse primer.
For primer concentration follows the requirement of your sequencing service.
If your insert is not too large, you could consider using standard primers (T7, M13, SP6, etc.) if your insert is in a compatible expression plasmid. Most sequencing service provides them at no extra cost.
Primer3 that is open access should be the best tool for you. You past your consensus sequence and ask the program to design primers for you. Its very easy to use.
Here is the link http://www.ncbi.nlm.nih.gov/guide/howto/design-pcr-primers/