Designing species specific primers for E.coli - please help? (Jun/13/2006 )

Hello! I'm supossed to design some species specific primers for E.coli for PCR.

Okay i do know the requirements for a primer, like no runs of more than 3 consecutive G's in either the primer or the probe, no G on the 5' of the probe.. etc. But the thing is how do i go about actually deciding which region i want to probe and prime? Sorry, but i'm really confused.

I've read a couple of journals and many prime and probe the 16s r DNA. also i know i must carry out multiple alignment with other bacteria strains (eg. S. aureus..etc) to identify regions of identity within 16s r DNA. but how do i go about doing that?

I'm not sure if i put this question in the right section though. It's a mix of bioinformatics and molecular bio. But please help! i'm very lost.

hmm...I would design a handful of rDNA primers and run them through Blast till I found a good one...but I bet there's an easier way. I'm going to move this over to bioinformatics; hopefully those crazy gurus can give you an easy way

Thanks! but how do you identify the region of identity? why do you need to blast the primers? isn't blast used to find out the identity or similarity of a particular sequence? anyone? please help! bioinformatics is completely new to me! thanks in advance.

Try "Design Primers" from BioInfoMan (http://bioinfoman.com ). It has a very cool tool to design primers automatically.

There are some of other unique tools from this program.

o Easy-to-use (the easiest!) tool to search and retrieve molecules from on-line databases.
o Powerful and foolproof cloning wizard: it also includes the gene synthetic cloning, TA cloning, D-TOPO cloning and Gateway cloning that are not available in any commercial products.
o One-click to design sequencing and PCR primers.
o Rare codon analysis of a sequence for any species.
o Optimized reverse translation:
• Codon optimization for any species
• GC content optimization
• Restriction site optimization
• RNA secondary structure optimization.
o Smart sequencing result analysis:
• Automatically trims the ambiguous sequences off the two ends of your results
• Automatically align reverse-complementary sequences
• Highlights the differences after alignment
• Aligns multiple sequencing results against a single target sequence and multiple input file formats are supported
• You can store your target sequences on-line (retrieved from GenBank or entered by yourself) and access them at any time.
o Auto-annotate constructs: By paste just sequences, the tool predicts all common features and converts your sequences to fully annotated GenBank format.
o Construct manipulation without losing annotation!
o Complete restriction digestion analysis.
o All types of protein and DNA sequence format conversion and manipulation: Users can do almost anything to manipulate the sequences, calculate the properties and so on.