design primers with custom end - To clone my gene into the MCS as fusions to the GFP tag (Mar/09/2008 )

i have designed primers with restriction sites of Xho-1 at 5' end and Hind III at 3' end. I want to clone a gene in same reading frame as GFP tag, how do i make sure that my gene is in same reading frame ?

I have added 2 nucleotides before the start codon, so my primer reads as :: XXXXXXXrestriction site-XX-ATG...cDNA.. (as the enzyme will cut after 1st base and so to complete the triplet codon "XXX", followed my my gene start codon, ATG).

I have to make 20 such constructs...any link or tools available to design such primers will be helpful.
Thanks !

Do you have an app like MacVector, or equivalent? If so, you can "insert" your gene into the plasmid, test the translation and modify the primers accordingly. Failing that, you could do the exercise on paper.

1. Check the reading frame from the plasmid.
2. Make your XhoI and HindIII cuts, then "ligate" in your sequence.
3. Check the reading frame, and you can add bases as required to restore the frame if it's out.
4. GET SOMEONE ELSE TO CHECK YOUR CALCULATIONS!!!!! You will feel really silly if you miss a frame-shift, and you have to go an re-synthesise and re-amplify, or do some mutagenesis/insertion expts (Trust me, two people in my lab have recently done that very thing... including the boss!!)

hello
I do have vector NTI in my lab, but im unaware of how the above mentioned analysis can be carried out using vector NTI.