Hi everyone! I performed my very first Gibson assembly (1 vector and 2 fragments) using the NEB Gibson Assembly Cloning Kit (#E5510S) and the assembly efficiency was quite disappointing as revealed by agarose gel electrophoresis. In general, what I’ve seen was that both positive (incubated) and negative (non-incubated) assemblies look the same, which tells me that the assembly efficiency was very low. So, I was wondering if anyone here has experienced the same issue and might be able to give me some “pro-tips” on how to make it work better.
Attached is an overview figure of my experiment and outcomes for reference. Below are the details of the experiment:
(a) On vector preparation: The vector was linearized using double digestion with NcoI-HF (NEB, #R3193S) and EagI-HF (NEB, #R3505S). Both REs display 100% activity in rCutSmart buffer and are not sensitive to procaryotic methylation (i.e., dam or dcm). Reactions were assembly with 1 ug of DNA and 20 U of each enzyme in a 50 ul final volume. Digestions were carried out at 37˚C overnight, following gel verification of successful digestion (final length = 3,920 bp). The digestion was gel purified using the QIAquick PCR & Gel Cleanup Kit (Qiagen, #28506) and eluted in nuclease-free water.
(b) On inserts preparation: insert 1 (494 bp) and insert 2 (2,090 bp) were PCR amplified using primers generating ~40 bp overlapping between fragments (Figure 1a). Amplifications were performed with Q5 High-Fidelity DNA Polymerase (NEB, # M0491S), and gel purified using the QIAquick PCR & Gel Cleanup Kit (Qiagen, #28506) and eluted in nuclease-free water.
(c) On molarity calculations: All concentrations (Figure 1b) were estimated using 2 ul of purified fragments in a Qubit v.2 (Invitrogen). NEB recommends 50-to-100 ng of a vector with a 2-to-3-fold molar excess of each insert (I also have seen some people recommending a 1:1 ratio, do you think it would be better?). I decided to use 60 ng of the vector, and used the following formula to calculate the vector amount (pmols):
pmols = (weight in ng) * 1,000 / (length in bp * 650 Daltons)
Therefore:
pmols = 60 ng * 1,000 / (3,920 bp * 650 Da)
pmols = 0.023
Next, I calculated the necessary mass in ng of each insert for a 1:3 (vector : insert) molar ratio (0.023 pmols of the vector, and 0.07 pmols of the inserts) using the following formula:
(Insert length in Kb / vector length in Kb) * (insert ratio / vector ratio) = insert weight in ng / vector weight in ng
Therefore, for insert 1:
(0.494 Kb / 3.920 Kb) * (3 / 1) = X / 60 ng
0.126 * 3 = X / 60
0.378 * 60 = X
X = 22.6 ng (which gives me 0.07 pmols)
And insert 2:
(2.089 / 3.920) * (3 / 1) = X / 60
X = 96 ng (which gives me 0.07 pmols)
(d) On Gibson assembly: No secrets here, just followed NEB recommendations: Mixed 10 ul of my fragments (see Figure 1b for individual quantities) with 10 ul of 2x Gibson Assembly Master Mix, for a final volume of 20 ul. Reactions were mixed by pipetting and incubated in a thermocycler at 50˚C for 60 min (NEB says that this can be increased to up to 4 hr, have anyone tested this yet?). For control, I used 10 ul of NEBuilder positive control (supplied with the Gibson kit) in a 20 ul reaction. Finally, I included 2 negative controls consisting of my assembly and the NEB control assembly without incubation (left at room temperature ~25˚C during the experiment).
(e) On outcomes (why efficiency so low?): After incubation, I ran 8 ul (equivalent to ~24 ng of the vector) of each positive (mine and NEB positive incubated reactions) and negative (non-incubation reactions) assemblies in a 1% agarose gel (Figure 1c). The result was very disappointing: both positive (incubated) and negative (non-incubated) assemblies look the same, which tells me that the assembly efficiency was very low. Breaking down the bands I can see that:
Experiment: band #1 is the expected size of [vector + insert 1 + insert 2] (6.5Kb), and band #3 of [insert 1 + insert 2] (2.6Kb). I also can see lots of vectors and insert leftovers (bands #2, #4, #5), meaning that the assembly was not efficient, or the molarity ratio should be improved. But most importantly, the incubation (50˚C for 60 min) seems to have little or no impact on the assembly efficiency: What is wrong with that? Because of these results, I decided to not keep going further.
Positive control: As for the NEBbuilder positive control, the bands are very weak in the gel, but I can see a shift in size (from ~3kb to ~3.5Kb) between positive and negative controls (see shift marked as white arrows in Figure 1c). So, I decided to keep going and used 2 ul of the assembly to chemically transform NEB 5-alpha Competent E. coli(High Efficiency) cells (provided with the kit), following NEB protocol. I plated 100 ul of the overgrowth (37˚C for 1 hr) and incubated the plate at 37˚C overnight. The next morning, I could see some colonies, but not many (~27). Do you think the transformation can be improved by using more volume of the assembly (~10 ul) and overgrowth the bacteria for 2 hr instead?
I really appreciate any comments on these results, which might help others in the future. Thanks in advance for your patience and help.
Dani.
Niels, thank you for your comment.
You're right about leaving the reaction at RT; the assembly might still happen. It's just weird that incubation and non-incubation led to the same results. I tried this week (to make sure it wasn't something wrong with me) but kept the negative control (all reagents + fragments) at the fridge (4˚C). This time I don't see bands #1 (full assembly) and #3 (insert 1 + insert 2) in the control, confirming that these bands indeed represent my assembly (partially and/or complete). Yes, I'm sure band #2 is vector-only because of its size (~4Kb, while full assembly would be 6.5Kb), and also because I see the same band in the new negative control.
I agree, even a faint product would be enough for transformation, and probably I'm overthinking this. Yet, I wish I could see more assembled products in the gel. It would give me more confidence when selecting colonies.
This week I will try assembling again, but this time I will ignore the 1:3 (vector:insert) molarity ratio and use 100ng of each vector and inserts. I might also be able to perform an assembly using the NEBuilder HiFi kit (maybe trying the 1:3 ratio and all fragments at 100ng), which will give me a side-by-side comparisson between these two kits and methods.
I will try and keep this question updated!
- Dani.
Just an update for those following this question:
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/
Thank you so much Daniel for your updates. I´m designing my primers for my first PCR -GBA but I would like to know if you are using the complete sequence of the primer + overlap region or only primer sequence ? Also I would like to know in your GA , there is a 3 inserts counting the vector or two. In my situation I want two inserts and the vector, that would be a total of 3 inserts ?
Hi Onias,
My assembly was 2 inserts + 1 vector, so 3 fragments counting the vector, same as yours. I would recommend making the assembly using a 1:1 or 1:2 (vector: insert) molarity ratio for a total of 0.1 - 0.2 pmol.
I would also recommend using the HiFi assembly kit; I performed a side-by-side comparison between HiFi and GB, and the HiFi kit is way superior to GB.
I'm sorry, but I don't really understand your question about the primers. Do you want to know if I'm using the primer + overlap or only the primer sequence to do what?
Please feel free to make any other questions.
Best of luck,
- Dani.
Hi, Daniel, thank for your reply. How are you doing ? I would like to ask you about primer design. Im worried about dimer formation. Where did you design yours ? i don't know to much about TD PCR...
Hi Onias,
The primers are a little bit tricky, cause sometimes there is not much space to design them. In general, you want primers 18 - 20 bp long, with a TM between 55 - 60 ˚C (this is the sequence-specific region), and an overlapping region of 30 - 40 bp and a TM > 48˚C (usually the overlapping TMs are much higher than this; ~60 - 70 ˚C). I use a combination of tools to design them, including:
SnapGene (really good for designing your cloning strategy, as it allows you to simulate the experiment): https://www.snapgene.com/
PrimerBlast is really good, it uses the primer3 engine to find primers: https://www.ncbi.nlm.nih.gov/tools/primer-blast/
Then I evaluate the sequences for hairpins and dimers using IDT Oligo Analyzer tool: https://www.idtdna.com/pages/tools/oligoanalyzer?returnurl=%2Fcalc%2Fanalyzer
TD-PCR is not that complicated. In general, you run your PCR for 10 cycles with your TM + 10˚C and -1 ˚C every cycle. So your desired TM is reached after 10 cycles. After that, you run another 25 cycles using the primer TM. Here is the paper: https://www.nature.com/articles/nprot.2008.133
Some tips: (1) make sure to use a high fidelity polymerase for your PCRs (such as Q5 from NEB); (2) if you need to purify fragments from the gel (for instance to get rid of dimers or off-target amplification), use the QIAquick gel purification kit followed by an extra purification step with QIAquick PCR purification. Make sure the 260/230 ratio of your purified fragments is > 2.0 (THIS IS CRUCIAL).
Best luck,
- Dani
Thank you so much for your replies Dani. I got another question. I read incubation must happens at 50 °C ; do matter the overlapping region with Tm between 60-70°C ?
Yes, the incubation should be carried out at 50 ˚C for 1hr. All TMs of my overlappings (40 bp) were around 75 ˚C, so don't worry too much about it.
- Hi Dani how are you ? Thank you for your reply. Im desgning primers for Gibson Assembly . My main query about that is the harpain and selfdimer formation ? Did you have problems with that ? My primers have high propability to form selfdimer . What can I do ? I´m desperate because I feel like I´ve spent lot of time designing primers.
Hi Onias, don't be desperate, designing experiments take time, and sometimes it can be frustrating, but don't give up. Some tips:
- Play around with the sequences: Check where the hairpins and self-ligation occur in your primers. Sometimes you can get rid of them by just deleting some 5' or 3' bases of your primer. For instance, you can go for a short target-specific sequence (~18nt) and/or a short overlapping sequence (20 - 40nt). I've never used overlapping less than 40nt, but I think you might be ok with 20 - 30nts. I use OligoAnalyzer Tool from IDT to check secondary structures in my primers.
- Just try on PCRs: Just order a couple of primers and do PCRs using different annealing temperatures or try the touch-down PCR that we already discussed before. Sometimes the primers look bad in theory, but in practice, they work just fine. Don't forget to assembly your reactions on ice and transfer them to a pre-heated thermocycler already set to the denaturing temperature. In my experience, it's better to invest some time optimizing the PCRs before going any further. There are so many parameters that you can change here (e.g., use the minimum amount of primer (0.05 - 0.1 uM), adjust input DNA, use less dNTPs, short annealing and extension times, less Mg2+ ...). Also, remember to optimize your reactions using a high fidelity polymerase (e.g., Q5 from NEB), including 10% of BSA and GC enhancer might also help.
- Isolate amplicon from the gel: After optimizing the PCRs, if you still see a band around ~100 bp (probably dimers) I would recommend excising and purifying the target band from the gel. Now, you want to make sure your purification is clean enough to proceed with the cloning. Your purified fragment should have a 260/230 ratio > 2.0 (this is critical). I'm using the QIAquick gel and PCR purification kit for that. You will lose a lot of your starting material, but you should have enough purified PCR product to continue until the end. I perform two consecutive purifications: first using the gel extraction protocol, then I purified the first purification product again using the PCR clean-up protocol (this will ensure that you remove all guanidine used in the first purification to dissolve the gel). For both steps, I wash the filter twice for 5 min. Verify the quality in a gel and nanodrop (or similar). A 260/230 ratio < 2.0 probably means that some guanidine is still present in your purifications, and that will most certainly block your assembly.
- HiFi no Gibson: Last, I would recommend using the NEB HiFi DNA assembly kit instead of the Gibson one. I made a side-by-side comparison of both kits, and the HiFi is waaaaaaaaaaaay more superior. You can ask NEB to send you a sample kit (enough for 10 reactions).
I believe we've covered everything now. Please, review the previous answers here. Hope this can help you. Sometimes molecular biology is just trying and error, but it will work eventually. There are lots of ways to circumvent bad primers. Good luck. - Dani.
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