my plasmid is pGL3-U6-... and sticky ends as gttttagagctagaaatagc. but the are not being joined. what to do?

[Muhammad Farhab]

my plasmid is  pGL3-U6-... and sticky ends as gttttagagctagaaatagc. but the are not being joined. what to do?

[Dr. Steven R. Bischoff]

Hi Muhammad,

I used UNAfold, and it does appear your sticky ends can form a hairpin deltaG = -2.23 with Tm 60C. If you are trying to assemble by isothermal/Gibson procedure, we have tested our eponymous cloning mix, namely NovoHelix HiFi DNA Assembly Master Mix, up to 65C and gotten assemblies by simply ramping the temperature from 50C to 65C. Our DNA assembly mix is not identical to Telesis Bio or NEB, so I don't know how those reaction mixes will perform with higher temperatures (65C). https://www.novohelix.com/Products/cloning-and-dna-modifying-enzymes

Additionally, we have tested our DNA assembly mix with high GC-content ssDNA sticky ends, and gotten successful assemblies with the NovoHelix HiFi DNA Assembly Master Mix and spiking in PyroPozzi, an extremely thermostable single-stranded DNA binding protein. Please check out the Rabe & Cepko preprint on the addition of ET-SSB in DNA assembly mixes. Again, PyroPozzi® is an extremely thermostable single-stranded DNA binding protein (ET SSB) isolated from a hyperthermophilic microorganism near Pozzuoli, Italy. The protein has been improved by directed evolution and rational design and remains fully active after incubation at 100°C for 12 hours. Due to its extreme thermostability, PyroPozzi® can be used in applications that require extremely high-temperature conditions, such as nucleic acid amplification by PCR and Gibson assembly. https://www.novohelix.com/Products/pyropozzi

A Simple Enhancement for Gibson Isothermal Assembly
Brian A Rabe, Constance Cepko
bioRxiv 2020.06.14.150979; doi: https://doi.org/10.1101/2020.06.14.150979

Conflict of interest: NovoHelix sells the recommended products, and I am the chief scientific officer of the company.

Wishing you the best,

Steve