Goal: I'm trying to create a concatemer of multiple short DNA fragments.
Background: I PCR amplified many sequences (112bp), each with two BsaI cut sites. There is a fragment within each sequence I'm working with, flanked by the two cut sites. I am using golden gate cloning to cut and re-ligate many of those fragments at the cut sites and assemble them into one long (linear) concatemer.
Problem: When I do the golden gate cloning protocol and run the product on a gel, the products are short. The largest one is generally shorter than 112bp, suggesting that the digestion is working but the ligation is not (see attached gel image).
Question: I am wondering if there's a chance the DNA is self-ligating at the cut sites to create a potentially circularized product. If that is happening, would it explain why I get no bands larger than 100bp?
What I've tried so far:
- 20 vs. 10 µl reaction sizes
- different lengths of digestion/ligation time steps during cycling
- 60 vs. 30 cycles
- Different input amounts (100ng, 10ng, 1ng)
If anyone has any advice or insight that would be greatly appreciated! Thanks!
the fig is from the golden gate cloning? I am not sure you could find the assembled fragment by running gel, you might try to run the PCV to see whether you could get it, or clone it to a vector.
Since your fragments are flanked by two identical restriction sites that can relegate together, self-ligation generating a circular monomer will be the favored outcome of the ligation. The way to increase the formation of concatemers would be to raise the concentration of the fragments.
Of course, this is a case of self-ligation. Not only this, you shall also face a problem with orientation. There is no other way, but to use higher insert concentrations, and thorough screening of the colonies obtained using PCR and Sanger sequencing.
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