I'm assembling one PCR fragment (4.2 kb) with my vector, pECFP (4.8 kb), and transforming them into NEB 5-alpha Competent E. coli cells. Both the fragment and the vector were purified using the gel extraction method. I have followed the instructions of the kit, trying different ratios of vector to fragment (1:1, 1:2, and 1:5) and allowing the reaction to proceed for 60 minutes or more, although there is only one fragment.
For transformation, I used 2 μl and 4 μl with 50 μl of NEB 5-alpha Competent E. coli cells for a heat transformation and then streaked the transformants on Kao plates (100 μg/μl). However, most of the time, I observe no colonies growing the next day. On the rare occasions when colonies do appear (only 7), they all contain the vector.
My next step was running 5 μl of the Gibson assembly product on an agarose gel to confirm the presence of the expected band (8.8 kb), and indeed, it was present. Despite trying various conditions, I consistently obtained a faint band, insufficient for successful colony formation.
Do you have any ideas on how I can improve my assembly and obtain the desired product? I would be very grateful, as I have exhausted my current strategies.
Thank you very much :)
Hi, Nuri!
Did you perform the ligation and transformation using the puc plasmid from kit control?
Apparently, the problem is in the transformation since you see the complete plasmid band. Did you perform the recovery step after the transformation?
What is the concentration of the backbone vector used for the ligation?
Hi, Flávia.
Yes, I performed the ligation and transformation with the puc plasmid from the kit, and everything was fine. I got colonies.
The concentration of the vector was within the pmoles (0.02-0.5) recommended in the kit. I used 0.025, 0.075, and 0.1 pM, and in ng, I used between 79, 235, and 315 ng, respectively.
However, after performing mini preps, and enzimatic digestions, I realized I only got vector but not my plasmid product.
As you said, the problem is in the transformation. I don't know if I should use more than 4 μl of the Gibson reaction (20μl) to transform 50ul of bacteria.
This is strange....
Its backbone concentration is high, 4ul would be enough for the transformation. You can even try to increase the amount to transform, but this may not be the problem.
I once read in a discussion here that the gibson kit buffer is toxic to some bacteria, but as you used NEB 5 this is unlikely since your control was fine. I personally don't like NEB5 very much, I prefer to use other strains like NEB Stable which bring better results.
Well, what I would try at this point would be: changing the bacteria, I would try using electroporation and reducing the antibiotic concentration of the selection medium enough so that the negative control does not grow.
How about using PCR to incorporate your insert into the plasmid? Something like here:
Article Overlap Extension PCR Cloning: A Simple and Reliable Way to ...
Try a control PCR on your gibson assembly reaction using vector primers to be sure you do indeed have the correctly assembled fragments. Sometimes it is necessary to use a different E. Coli strain. Try NEB stable or any other E. Coli strain you have available. Occassionally we have needed to use electroporation for hard to clone fragments.
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