Sorry another Q: when I calculate the TM I do not consider the insert that does not have correspondance within my seguence?
Yes, quiet right. Only the DNA sequence that actually binds to the template is used for the tm calculations. If primer is 100bp long and 23bp anneals to the template. The tm calculated on the 23bp.
Also you might want to use the enzyme XmaI rather than SmaI for the digest. Both restriction enzymes cut the same DNA sequence, but XmaI has two advantages over SmaI. XmaI 5' overhang while SmaI is blunt end. Thus the ligation is easier. Next XmaI cuts at 37 C while SmaI at 25 C. Thus you can use XmaI and BamHI in a double digest in NEB buffer 4.
Thank your for answer
Things aren't simple though:
I have multiple isoforms(different splice variants)
2 have no site for SmaI within the site but have for BamH1
6 have sites for Sma1 and BamH1
So for the first two I was thinking of using EcoR1 at 5' end and SmaI ( or now XmaI), and I shell put overhanging nucleotides (A,T) 1-2 on both sites as I can cut and not clive my gene of interest, or shoul I put more?
But for the other 6 I guess I must use Sma1 at 3' end and add no overhanging nucleotide(?) so I wouldn't need to clean it with Sma1 at that end, otherwise cuts my gene-so I would prefer blunt end.
What do you think?
PS: so when calculatingTM, I even do not consider the restriction site that I insert in my primer, corret?
Thank you for your help:)