I usually do 20 bp overlaps but I don't see why 40 would be bad. It might even be more efficient than 20. Gibson Assembly isn't temperature cycled like PCR, I think the annealing temperature of the overlaps is only a problem if it's significantly lower than 50°.

There's always a higher chance of longer primers misbehaving in PCR itself, but they might work with no issues.

Thanks, Alexandra. It's the first time I'm doing this, so a lot of noob doubts.

You need to ensure Tm of overlaps higher than the incubation temperature 50C. Circumvent secondary structure in the overlaps. I usually design a overlap of 30bp and have not seen any mistake. There is a paper for the optimization of Gibson Assembly that you can refer; doi:10.1038/nmeth.1318.

Thanks Qing, I will take a look at it.

Hi, I have a similar problem. I designed primers for Gibson assembly with 60 bp overlaps between adjacent fragments, I need to assemble 2 fragments of DNA (2800pb and 1072 pb; 4099 and 1072pb) and the plasmid (7300 pb). I tried gibson assembly to 50°C, like the manufactor instructions and nothing. I observed the product of assembly by agarose gel and I saw individual fragments and a little cloud of the "assembly hoped". Do you think that I need to probe more temperatures? I garantice that I have the concentrations of fragments need. I read that the plasmid digest maybe can recirculariced and in my agarose gel I saw a intense band from the plasmid, and I considerated decrese this concentration (

Hi Laura,

60 bp sounds like a lot to me (but the method is also new to me), is that recommended by the manual? Below is one of the references that I found when I was designing my primers. According to that, the recommended overlaps should be around 40 bp.

Incubation time is also a key point. NEB says that "For assembling 2–3 fragments, 15 minute incubation times are sufficient", but also "Extended incubation times (up to 4 hours) have been shown to improve assembly efficiencies in some cases", so I guess you can play with that parameter a little.

Lastly, the quantities are another thing to consider: "NEB recommends a total of 0.02–0.5 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector (...) Efficiency of assembly decreases as the number or length of fragments increases".

Hope someone with more experience can jump here and provide you with a better answer.

Cheers,

Dani.

Ref. to homologous arms (overlapping) length:

https://www.biocat.com/bc/files/Gibson_Guide_V2_101417_web_version_8.5_x_11_FINAL.pdf

Ref. NEB manual for Gibson assembly:

https://www.med.unc.edu/pharm/sondeklab/wp-content/uploads/sites/868/2019/10/gibson-cloning.pdf

Thanks for you answer. The overlaps have 38pb +22pb for the primer. I considerated the amount of each fragment and plasmid, I verified integrity and concentration, even I tried 4 h incubation. I decided to try the transformation. In a couple days I will know if its work.

Best regards,

Laura

Did you solve the issue, Laura?

Laura Espinosa , I start performing my Gibson assemblies and my outcomes (so far) look pretty much like your gel. My main hypothesis is that the overlaps have a too high TM, and therefore the assembling is not being facilitated. Did you solve your issue? Any recommendations?

Hello Daniel Fenando Paulo and Diego Orol Daniel. Its really frustrated. Even I preparated a Master mix homemade. I still don't have good results :/

Hi Laura,

My assembly finally worked. The key - for me - was using high-quality DNA fragments for the assembly reaction. Following are some lessons I learned:

- I found the NEBuilder HiFi DNA Assembly kit WAAAAAAAY superior to the Gibson kit. Maybe it might also be better than the homemade reagents, so it is worth a shot.

- What made all the difference was making sure my DNA fragments were as clean as possible (meaning an A260/230 ≥ 2.0 and A260/280 = 1.8 - 2.0). I found that carryover salts (such as guanidium from gel extraction) block the assembly reaction completely. So this is a 'MUST TO' step.

- Make sure your PCR amplifications are as specific as possible, resulting in only one strong band of the expected size and NONE primer-dimers. Dimers can heavily interfere with the assembly due to their overlapping regions. I had good results using touchdown-PCRs and a primer end-concentration of 0.1 uM. In my experience, it's worth it to invest time optimizing this step.

- However, if you need to purify bands from the gel (like I did), I recommend using the QIAquick Gel Extraction Kit followed by an extra purification step with the QIAquick PCR Purification Kit. That's the only way I found to get rid of the salts present in the gel dissolving buffer. Allow the washing buffer to sit in the column for 5 min before centrifugation during the gel cleaning step. Next, use two washing steps during the extra purification with the PCR kit. Be prepared to lose large amounts of DNA here. Also, if possible, cut your bands under a blue light instead of a UV light. That will help prevent damage to the DNA structure during gel extraction.

- I had success using 1:1 and 1:2 (vector: insert) molarity ratios for a total of 0.12 - 0.14 pmol. I used 60 ng of the vector in the 1:2 ratio. Always incubate the reaction for 60 min at 50 ˚C. NEB also says that in some cases incubating for 4 hrs might result in better assemblies. I think that might apply to complicate multi-fragment assemblies.

- You can verify the correct assembly by PCR. Dilute your final assembly 1:4 and use 1 ul in a 50 ul PCR reaction spanning your assembled fragments (I used M13 primers as my assembly was within the MCS of my vector). You should see a strong band with the expected size of your assembled fragments if the reaction worked. Other fragments (relative to closed vector, other unspecific assemblies, etc ...) might also be visible, but they shouldn't be more strong than your targeted amplification.

- I had good results transforming 50 ul of chemically competent cells (NEB 5-alpha) using 4 ul of the 1:4 assembly reaction. Outgrow your transformation in 950 ul of SOC for 90 min at 37˚C, and use 100 ul (half of this volume should be better) to plate. You should be able to see tons of colonies on the next day.

Well, hopefully, this can help you. This can be very frustrating, but if you are using high-quality DNA fragments and the HiFi kit, the assembly should work just fine. Otherwise, you might need to re-design your assembly strategy. One last tip, I think you might have more success assembling 2 - 3 fragments (including your vector) at a time.

Cheers, - Dani.

NEB HiFi:

https://international.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/nebuilder-hifi-dna-assembly

Touchdown PCR:

https://www.nature.com/articles/nprot.2008.133

QIAquick Gel Extraction Kit:

https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/dna-clean-up/qiaquick-gel-extraction-kit/

QIAquick PCR Purification Kit:

https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/dna-clean-up/qiaquick-pcr-purification-kit/

Daniel Fenando Paulo Hi, Daniel. I'm trying to set up my Gibson assembly protocol these days.

For asking a few questions, how did you design your primers and overlap length?

1. Did you use snapgene primer design tool as you first did?

2. How long was the primer overlaps?

3. How about the Tm of overlaps?