Hello,
I am trying to clone 1334 bp insert into 6.1 kb vector using HiFi DNA assmebly/Gibson assembly kit. I am taking 100ng of vector and 1:5 molar ratio of vector: insert and 4 hrs of ligation at 50 C. After transformation using NEB DH5 alpha competent cells I am not getting any colonies after overnight incubation. What could be the possible reason? How can i optimize reaction?
The first thing I noticed is that your insert to vector ratio is higher than what is recommended (2 insert:1 vector), and you're using an excessive incubation time at 50°. NEB recommends only a 1 hour incubation at most for complex assemblies with 4-6 fragments, and only 15 minutes for assembling 2-3 fragments. Gibson/NEBuilder Assembly reactions are mechanistically different than traditional ligations and using excessive insert to vector ratios and longer incubation times can be detrimental to proper assembly. I would first re-try your assembly using the recommended conditions.
If that doesn't work, then I would also try the positive control that comes with the NEBuilder kit:
(This is from the NEBuilder manual)
Q20. What should I do if my assembly reaction yields no colonies, a small number of colonies, or clones with the incorrect insert size following transformation into E. coli?
A20. Assemble and transform the NEBuilder Positive Control provided with the NEBuilder HiFi DNA Assembly Master Mix/Cloning Kit (see page 11, 12). Successful assembly with the NEBuilder Positive Control will demonstrate that the assembly mixture is functional and the transformation conditions are suitable.
Positive control reaction set up can be found here:
https://www.neb.com/en-us/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol
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