Hi everyone!
I'm trying to insert a ssDNA(~100bp) to my vector(~10000bp) by Gibson assembly. In my plan, the vector are digested by BamHI and NdeI overnight, gel purified and then incubated with ssDNA(1:10) in 50 ℃ for 1h.
I got plenty of colonies from my plate. But only about 50% of them have the correct insert. The strange thing is the rest of them are not the vector undigested. In fact, sequencing result shows that many basepairs near the enzyme site are deleted and the rest are ligated.
I also transformed and plated a 'control' with only digested vector in Gibson reaction. The strange thing is it had more colonies than my reaction group! I sequenced some of the colonies and got similar result: Basepairs near the enzyme site disappeared(missing lengths are highly variable, from 200bp to 6000bp)
As I'm trying to use the design to build a library, such stange and high backgrounds trouble me. Does anyone have ideas about why such thing happened and how to prevent it?
Digesting plasmids overnight might cause unwanted activity of unspecific nucleases. NEB for example have enzymes that can be used overnight but I never perform digestions overnight, with a couple of hours it is more than enough to have the plasmid completely digested. I think the deletions you have seen might be due to nucleases contaminants in your restriction enzymes.
E.coli strains for cloning (recA mutants) still retain capability for recombination and NHEJ in vivo. So that would explain why you get closed plasmids in your E.coli transformants. When performing the negative control transformation you are putting more selective pressure for the cells to close the linearized vector to be able to grow on selective media, while transforming the product of Gibson reaction, cells are being provided of closed vectors favoring those over the linearized vectors that required repairs.
Xi Jiang
Thanks for your advice.
I guess the recombination might be an important reason, but I'm not sure shorter digestion time helps. I've tried about 3h digestion during the troubleshooting, but got similar result.
Also, As the two enzyme sites are close in my vector, shorter digestion time led to more empty vector in my plates.
It is common to get background with gibson assembly. The reason is you have an exonuclease and ligase and dna polymerase in same mix (everything that is required to seal a plasmid), which happens in your case is that the exonuclease can digest to either turn this into a blunt ligation or simply expose regions that can anneal to each other and ligate (hence various size of deletion). You are also using ssDNA the efficiency will be lower since your are requiring the polymerase to fill in a larger fragment as opposed to what happens when you use dsDNA. I was suggest you order the complement to your ssDNA hybridize it then do the assembly and see if that improves it.
Hanna Alalam
Thank you for the advice.
If it still doesn't work, I might try to amplify my oligo... However, my ssDNA has random sequence so it might lead to other problems like skewing.
Finally, it worked when I used another backboen with XmaI and PacI sites...
But just for reference, a extension reaction conducted by High-Fidelity DNA polymerage that generates ds DNA oligo will increase the efficiency.
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