Dear cloning community,
Is it possible to use Overhangs with large Tm difference with NEB Builder HiFi DNA assembly?
5´ 26 bp Tm 57ºC
3´ 24 bp Tm 72ºC
Unfortunately, extending the 5´ doesn't help much because it is a high TA region, and the same goes for reducing the 3´(High GC region)
The temperature difference does not really matter for the actual assembly step as long as they're both 50ish degrees or greater, since that's the temperature the ligation/polymerase fill in reactions take place at.
Your problem here is not the Tm difference itself, but primarily the high GC arm. High-GC-regions mispair with other high GC regions, even if the precise sequence doesn't match. This causes the assembly success probability to go down.
You can try, but if it's not working, then you'll need to think about how you can get what you want while working only with GC/AT balanced overhangs.
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