Help designing primers; also help with viewing GenBank - (Jul/16/2008 )

I am PCRing a gene. My mentor told me to get the surrounding few nucleotides downstream and upstream from my gene of interest, but I'm not sure how to view them.

If using GenBank, Glucose dehydrogenase gene

I need to design a primer for that gene. I was planning on just going about 10 bps in from each end and starting with complementary bases. Then, I was going to include a restriction site on the 5' ends of the primers and a few extra bases after the recognition sites.

I'll calculate melting temps and adjust accordingly, but does the above sound advisable for my primers? I'm ligating the gene into a vector after PCR so that's why I need the restriction sites.

If that is an okay plan, then I need a way to view the bases up/downstream for my gene. Does anyone know how to do this in GenBank? If GenBank can't do it, what is another way to find out?

Thanks,
Alex

NC-002578 gives the whole genome sequence of your organism. You can follow the links to either get the fasta format of your genome and search for your sequence or find your gene in the genome viewer and go a couple bases upstream and down stream.....

Hope this helps!

Please note this is the correct accession number for ur gene
X59788
http://www.ncbi.nlm.nih.gov/entrez/viewer....e&val=48914
ACC no X59788

Your ORF of interest Extends from 349..1410

GAATTCTTTCAATGAAACCTACTTTTAGACATTTTTGTGAAGAATATGCACCGTAATCAGCATTTGATTT
ACTTGATCCATTGGATCGATTGTAAACGAACGATCCATGAAATCCGCGAGTCAAAATTTTCCACATCATT
ATTGGTTATGTAAACATCACAATAAATCCTTTTGATCCGATTTCACTTCTAGCAAAACATGCACCAGGAC
GCTGCTCAGGGACATCGTTTCCTTCATCCGTGCAATATGCGGGCATCATATCGAAGGCCAACATTGCGTC
CAATAAACGGTTCTCGAAATGCGCAACACATTAGAAATTAATTAATAGAATCTAGCATTCCCGCACTTAT
GACTGAACAGAAAGCCATTGTGACAGATGCGCCCAAAGGTGGCGTGAAATACACAACAATAGACATGCCT
GAACCGGAACATTACGACGCCAAGCTTTCACCTGTATACATAGGAATATGCGGAACGGATCGTGGAGAGG
TGGCTGGTGCCCTGTCATTCACGTACAATCCAGAGGGGGAGAATTTTCTTGTTCTCGGGCACGAGGCGCT
TCTGCGTGTTGACGATGCCCGTGATAATGGCTACATAAAGAAGGGCGATCTTGTAGTACCCCTCGTGAGA
AGGCCTGGAAAATGCATCAACTGCAGAATAGGCAGGCAGGATAACTGTTCCATAGGTGATCCGGACAAAC
ATGAGGCTGGAATAACTGGGCTTCATGGTTTCATGCGCGATGTCATATACGACGATATAGAGTATCTCGT
TAAGGTTGAAGATCCAGAACTGGGAAGGATCGCAGTGCTTACGGAGCCTCTGAAAAATGTCATGAAGGCT
TTTGAGGTCTTCGACGTTGTGTCAAAAAGATCCATATTCTTCGGGGACGATTCCACGCTCATAGGTAAGA
GGATGGTCATAATAGGCAGCGGGAGCGAGGCTTTCCTCTACTCGTTTGCAGGCGTAGATCGCGGTTTCGA
CGTCACAATGGTGAACAGGCACGATGAGACGGAAAACAAGCTGAAGATCATGGACGAGTTCGGCGTCAAG
TTCGCAAACTACCTTAAGGACATGCCGGAAAAGATAGATCTCCTGGTTGACACCAGTGGTGATCCAACGA
CGACATTCAAGTTCCTCAGGAAGGTAAACAACAACGGCGTCGTCATATTGTTCGGCACGAACGGCAAGGC
GCCCGGCTATCCAGTGGATGGCGAGGACATAGATTACATCGTGGAGAGGAACATAACAATAGCCGGATCG
GTTGATGCCGCGAAGATACACTACGTGCAGGCCCTTCAGTCCCTCAGCAACTGGAACAGAAGACACCCAG
ATGCCATGAAGAGCATCATAACATACGAGCGAAGCCGTCCGAAACCAACATATTCTTCCAGAAACCACAC
GGAGAGATAAAGACGGTGATAAAGTGGCAGTGAAGATCCTCAGGGGAGAGGAGATCGCCGAAAAGAAAGC
CGAAAACCTGCATGGGATAATTGAAAGATCCGGGCTCGAACCATCGCTGAAACTTATACAGATTGGAGAT
AATGAGGCTGCCTCAATATATGCCAGGGCAAAGATCAGAAGGGGAAAGAAGATCGGATAGCCGTGGACCT
GGAAAAGTATGATGACATTTCGATGAAAGACCTTCTGAAGAGGATAGACGATCTAGCGAAGGATCC


Your gene of interest (ORF) is 1062 bp. Start codon and stop codon for the gene are marked in bold
The 5' and 3' Flanking sequences are underlined and marked in blue. Try designing primers close to your start and stop codons. Hope this helps for your primer design.

Regards
Avinash

http://aviprem.gq.nu

I find the Kyoto Encyclopedia of Genes and Genomes (KEGG) database particularly helpful for such things, as it includes all kinds of information about any given gene, and provides clickable links to all manner of cross-referencing databases. The entry for your gene is here. In the NT seq window, you can specify the number of upstream and downstream bases you'd like to include, then click the "NT seq" button to get them.