Hey everyone,

 

I am an undergrad researcher that has been tasked with making new plasmid standards for qPCR on several different nitrogen fixing genes so i need to track down the exact sizes of each gene so i can verify the correct gene was amplified in the preliminary PCR reactions. I thought this would be a cakewalk but it turns out it is not. I'm finding that alot of primers test for genes by amplifying only a small portion of a conserved region of the gene and not the entire gene itself, so when i search for them i come up with several different sizes depending on what primers are being used.

 

The genes im working with are nitrogen fixing genes and a few others: nifH, nosZ, nirK, nirS, bacterial 16s, fungal 28s, Bacterial amoA, and Archaeal amoA. All i have available are the primer sequences for all the genes of interest. It seems like with those sequences i would be able to enter them into some database and get the information i need, but i am very unfamiliar with the various databases available for this sort of thing. Any suggestions would be greatly appreciated!

 

Thanks in advance,

 

Phil Colgan

 

Assuming that the genes are from sequenced organisms you can get the complete sequence e.g. from uniprot or from NCBI (using the nucleotide database).

 

Note that gel-based size confirmation can sometimes be insufficient. The best would obviously be sequencing the amplified products, but lacking time and funds a simple restriction analysis can give at least an additional layer of security.