Hi,

I think You can design primers manually by selecting the first and last 25 nucleotides of the 2Kb sequence of your gene which you want to amplify. But check the designed primers compatibility and for the formation of any secondary structures by inputting the primer sequences into some online tools like primer blast, multiple primer analyzer, primer3 etc.,

Hope this may help you....

Hi Javed

since you won't be able to sequence the 2000bp length of your fragment I advise you to cut it and design overlapping regions. if it's for sanger sequencing, around 500bp length for the new fragments should be enough. if it's for NGS, depending on your sequencer, I would approach the 200bp length. for my part I use Oligo7 to get the hand on some parameters of the primers, but you can also use the tool from EuroFins (https://www.eurofinsgenomics.eu/en/dna-rna-oligonucleotides/oligo-design-more.aspx) but never forget to verify them on some other site as the UCSC (http://genome.ucsc.edu/cgi-bin/hgPcr) to get sure you'll amplify selectively the only and right sequence.

fred

Dear Javed,

It's quite simple, try to follow these points:

- Check the sequence of your gene on the NCBI (https://www.ncbi.nlm.nih.gov/pubmed/) or ENSEMBL (https://www.ensembl.org/index.htmland) be careful about introns that usually are very huge.

- If possible "copy and paste" the region from 150 bp before the exon and 150 bp after it, on some primer design tool such as Primer3 (http://primer3.ut.ee/) or Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/).

These tools will give you several pairs of primers. I suggest you to choose the ones with Annealing Temperature around 60°C and 50 bp before and after the exon because you know that the first 30bp of the sequence it's always very "dirty" so in this way you'll be sure to have a clean sequence to read.

With the same primers you can perfom PCR and Sequencing for every exon.

2000 bp it's not too much to amplify. If problems you can easily overcome them dividing the region of your interest in different part be careful to overlap them.

Good Luck

Edoardo

There is also the option of designing internal primers only for sequening