16s primers and PCR - (Jan/17/2008 )
Read the post on 16s (http://www.protocol-online.org/forums/index.php?showtopic=33339) and find it helpful especially the link. I'm extracting the genomic DNA tomorrow and will be running PCR with these primers obtained from another lab:
60F: 5' tna nac atg caa gtc gak cg
1392R: 5' acg ggc ggt gtg trc
This will be my first PCR and will be running gradient to see which is a better temperature. I'm not sure if any of you guys here deals with that specific 16s primers (DNA or RNA?)
I'm not sure about some letters in the sequence (i.e. n, k, r) as I know DNA or RNA bases doesn't have such letters (Do I call this 16s rDNA or rRNA sequence analysis in my report later?)
Getting the PCR product, is there a need to clone or just sent it for sequencing?
Help much appreciated. Have a nice day people.
Check out the references here: http://openwetware.org/wiki/Bacterial_species_identification
The letters are the IUPAC codes for mixed bases.
N = aNy base (a t g or c)
K = Keto, (g or t)
R = puRine (g or a)
I call it rDNA, ribosomal DNA.
If you get a single band, and gel isolate it, you can send it directly for sequencing, assuming you know that the initial sample is pure. If you have a mixed culture, then the sequencing will give you a mess, and you need to clone the PCR fragments to isolate individual DNA strands and amplify them by growing a pure culture from a single plasmid molecule.