primer design from mRNA - (Mar/24/2008 )
WOW!!!!!!!!!!!
Thank you so much!
It's the first time i try to design primer and so i'm not expert!
I just haven't understood how you have seen the coding sequence and how you have seen the second exon's begining/end from mRNA sequence...
I have done how you said to design primer per NOP rat and i haven't had problems, cause i just used exon's 2 (257bp) and 3 (356bp) sequence (long intron) and i hope it's right! Then i have tried to design primer for rat GAPDH but i have had some problems... i have tried my target in NCBI but i was not able to find the gene in ensembl... what's wrong?! 
Thanks a lot again!!!
-ros75-
QUOTE (ros75 @ Apr 12 2008, 02:57 AM)
I just haven't understood how you have seen the coding sequence and how you have seen the second exon's begining/end from mRNA sequence...
I have done how you said to design primer per NOP rat and i haven't had problems, cause i just used exon's 2 (257bp) and 3 (356bp) sequence (long intron) and i hope it's right! Then i have tried to design primer for rat GAPDH but i have had some problems... i have tried my target in NCBI but i was not able to find the gene in ensembl... what's wrong?!
I have done how you said to design primer per NOP rat and i haven't had problems, cause i just used exon's 2 (257bp) and 3 (356bp) sequence (long intron) and i hope it's right! Then i have tried to design primer for rat GAPDH but i have had some problems... i have tried my target in NCBI but i was not able to find the gene in ensembl... what's wrong?!
I don't know where you search for the genes in NCBI, but it has a special search dropdown called Gene, where it's best to start. Usually you search for the name od the gene and species (like "Rattus norvegicus GAPDH"), and get a page like this one for Rat GAPDH. There is some serious info on this page. First in the Summary you can use a direct link to Ensembl or others (not in this case, the gene is still provisional in both databases and the link can change a lot and is not working now).
Next there is a section Genomic regions, transcripts, and products where you can acces mRNA or protein sequence or other linked material from NCBI, (if you mouseover the label "NM_017008+2" and click it, you get to mRNA reference sequence I talked about).
Important thing is also Genomic context section, where you see the chromosome location, and if you click the link "See Gapdh in MapViewer", you can get a set of maps you choose to (among them is one called ensGenes that is particulary usefull now).
Finding this gene in Ens was a bit tricky since there is one gene called GAPDH for rat that is predicted and located on chromozome 16, which is obviously not the one we're looking for. NCBI claims it on chromosome 4. How I find it in the end, was to enter the MapViewer for Gapdh gene and on the ensGenes map found it collocalise with something called "LOC685186" in Ensembl, it's even clickable.
Here is Rat GAPDH from Ensembl.
On Ensembl you can see where the coding sequence starts, because non-coding is marked yellow in the Transcript Info, same way you find beginnings/end of exons, odd and even are in different colouring.
You can't find it this way in NCBI, but you pick few bases from Ensembl exon beginning and let your browser do Search in page for that sequence in FASTA format and that's how you find it.
Next step in desining primers is checking them in BLAST, to see if the binding is specific, if it really catches your desired sequence and so.
Also if you have time, go through the Help section of Entrez, it has some cool video tours to help beginners.
-Trof-
TROF!!!!!!
Thank you sooooooooo much for your helpfull advices, the first step is done!
-ros75-