For desigining yeast genotyping primers, I often use SGD Primers (for yeast), or UGENE. I haven't used NCBI Primer Blast much yet but hear it's also great. Before adding restriction enzyme cut sites onto the ends of primers for cloning, check out whether your enzymes can cut near the ends of DNA fragments on New England Biolabs website.
Verification Primer Design
There are multiple ways in which you can PCR verify following a transformation. If you deleted a gene with a selectable marker gene such as URA3 for example, you can either design primers external to the sequence that was modified or use a single primer that is internal to your substitution cassette URA3 and an external one. If you plan to sequence your PCR amplicon, you will need to use two external primers. If you choose the two external primer route, your PCR products must have different lengths. For instance, you cannot delete a gene nearly the same length as URA3 with URA3 and verify with external primers as both the positive and negative result will yield the same length PCR product. For this reason the "one internal" route is a good alternative- you will recieve a PCR product only if your transformation was successful, however the absense of a product could be indicative of an unsuccessful transformation -OR- PCR conditions. That said, primers are cheap and if you are determined that your transformation be found successful quickly, you may want to order three primers- two external and one internal- in case one method of verification fails.
1. Download your locus sequence (+/- 1 kb) from SGD, and paste it into your favorite sequence manipulation program such as Gene Construction Kit.
2. For external verification choose forward and reverse sequences outside of the manipulated region, internal primers can be either forward or reverse depending on how you pair it.
3. Forward primers will have the same sequence as the coding strand (since they will anneal to the sense strand), while reverse primers must be the reverse compliment of the coding strand (since they will anneal to the coding strand). To paste the reverse compliment in GCK, copy the reverse primer sequence off the coding strand, open a new file and Special Paste the "Inverted Sequence." You should see that the 3' end of the primer sequence should compliment the 5' end of the region as it appears in the locus file.
4. Verfication primers need only be 20 +/- 1 bp. Copy prospective primer sequences into the Oligo Tm Calculator and find pairs which differ by less than 2 degrees. Follow that by using mFold to determine if there are any secondary structures which melting points near the annealing temperature. Change [Na+] to 50 mM and [Mg++]= 2 mM and leave all the other parameters alone. After folding, click on the "Thermodynamic Details" of all the possible secondary structures and make sure their melting temps are less than 45 degrees.
5. Last but not least follow the BLAST procedure in Primers to determine if your primers will anneal elsewhere in the genome.
6. Go to the IDT website and order your primers. Shipping usually takes 2-3 days.