More of a bioinformatics question

[PhilC]

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

 

[CharonY]

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.