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Adding tag using overlapping PCR - (Mar/05/2013 )

I wish to add an HA tag and 2 restriction sites in the N ter of my protein. all together it is a long primer (too expensive since I with to do that to more than one protein...)
I have 2 questions -
1) can I break the long primer to 2 smaler ones with overlapping area and do it all in one PCR reaction? if so, how much overlap do I need? how do I calculate the Tm?
2) if I have to do 2 PCR reactions- I know I have to calculate the Tm only considering the part of the primer that matchs the gene, however after few rounds all the primer (including my addition) will anneal (and i will probably be higher Tm). should I change the Tm after few cycles?
Thanks ahead !!! I really need help!


You can also consider a gene synthesis approach. These days it costs $150 for up to 500bp. If your HA nucleotides and your start codon come together to create a novel restriction site, then you can cut and paste this new 5'-end into your other constructs.


Thanks. I will think about that.