BLAST Help , Designing primers... - (Sep/19/2008 )
I am new to BLAST , I dont know how to use BLAST. I am designing primers for qPCR, Now i have some primer pairs i dont know which one to choose based on BLAST , the nucleotide BLAST gives some results based on E value , Query coverage, total score, max ident...i dont know what are these things, so i attached some screen shots of BLAST results for 2 forward primers , Please explain me which one to choose and why ..and what should be values for the good primers....
based on blast, I do not think you can determine which one is better. The results only tell you where it is located, whether queried primers are specific or not.
If you want to know which one is better, you have to do other analyses after blast, which include dimer, hairpin et al.
once you have used BLAST to determine specificity, I would recommend putting your primer sequences into the search tool above. it will help you look for secondary structures that can cause problems, as well as give you information regarding melting and annealing temps, etc. that you will need for your PCR program.
I already designed primers which does not form any primer dimer , cross dimer, false priming and hairpin structures..so check my screenshot..the first one with 100% is my target template...so these 2 different forward primer give 2 different results...which one would you choose..?
Either one you can choose
NCBI has recently released a tool to design primers interfaced with blast, so you can do both the things from the same interface.
Maybe you can have a look at it: