Details of the experiment:
We aim to clone 3 gene segments into a vector.
Details of the gene segments:
- The first segment has a concentration of 34 ng/µL and is 1445 base pairs long.
- The second segment has a concentration of 30 ng/µL and is 125 base pairs long.
- The third segment, is 4758 base pairs long, with a concentration of 150 ng/µL, and is a PCR product.
These segments were designed to have an overlap of approximately 30 nucleotides with each other.
The vector has a concentration of 29 ng/µL and is 1850 base pairs long.
I have repeated this reaction using various volumes, but sometimes I did not observe any colonies, and other times, when I sent the colonies for Sanger sequencing, I found that one fragment had been inserted while the other two were not, resulting in different combinations.
In the last reaction, I used the volumes recommended by the NEBuilder site:
- Vector: 4.5 µL
- First fragment: 3 µL
- Second fragment: 0.3 µL
- Third fragment: 2.2 µL
- NEBuilder HiFi DNA Assembly Master Mix: 10 µL
- Each are = 0.114 pmoles
I incubated the reaction at 50°C for 5 hours, then performed a chemical transformation into E. coli Stbl3 strain.
I only observed 2 colonies, but after performing enzyme treatment and sending sanger sequencing, I saw that the desired segments were not inserted.
Could you help me understand what the problem might be?
You will get much lower cloning efficiency if the overlaps are 30 nucleotides. You should aim for an overlap of 15-20 nucleotides; the shorter the overlap the better, as long as it has a melting temperature >48°C.
I've never done Gibson assembly where one of the inserts is ~3 times bigger than the vector, but it should still work. I use 50 ng of vector, and then use 3:1 insert:vector molar ratio. You could try these amounts instead. For your small insert, you might want to use 5:1; this is recommended by NEB.
Make sure you have checked all of your fragments and your digested vector on a gel. Make sure all your DNA is good quality. You can check the fidelity of your Gibson assembly mix using the positive control that comes with the kit. If all of these checks look good, then the problem is the design of your overlaps (in my experience, overlap design is the cause of poor Gibson assemblies >80% of the time).
You'll want to also increase the relative concentration of the longest fragments relative to the others. Basically, the larger the molecule, the smaller the ratio of the "sticky ends/overlap" compared to the size of the molecule. I know it sounds counter-intuitive, but you want MORE copies of the larger molecules compared to smaller to increase the likelihood that the ends interact.
Good luck!
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