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Primer design - Primer design for PCR with TE sites (Jun/13/2008 )

I need a help.
I am designing primers to amplify a 10 kb region, (5kb upstream form Exon2 and 5kb downstream of exon 2), which needs addition of restriction sites to fwd and rew primers.
I have come up with primers as
5' - ATCG GGTACC ATG(start) .....complimentary bases
3' - AAGCT GCGGCCGC TTA(stop)...
Do they look ok.
My question was, do I need to incorporate start and stop codon ATG and TTA to the primer, or find a site like say NNNNATG in the mRNA of mine.And then design the primer from there on.
Also does the clamp sequence goes first, then RE site and then the ATG for fwd primer. Similarly does the rew primer needs to be cap seq, RE and then TTA???
I am using the vector pcDNA as it has the same RE sites of Acc65I and NotI in the MCS region.
This is the first time I am designing primers. So any help or advice will be helpful.


Hi Friend,
I am not sure about you but look at this site.
It may help you!


I am not sure what is your purpose for the cloing, to express your gene? Why only amplify exon 2, why include 5 kb up and down exon 2?