Hi,

yes, the transformation efficiency decreases with plasmid size.

First of all, you may try electroporation

Then you may use slightly slower grow condotions, likely 25...30C.

You may try rising Mg2+ and/or Mn2+ in your heat shock transformation buffer.

You may use positively charged peptides (like lactoferricin B or so) to aid plasmid with neutralized surface charge to enter the cell (there are som works on cell penetrating peptides, but I don't know whether synthesis or purchase is an option for you). In this case you may first mix plasmid and a peptide in mQ at different ratios, then add 2...3volumes of bacterial suspension and leave at 20...25C for 10...60min, then spin, resuspend pellet, plate.

Of course, in all the cases there will be a need for titrations etc.

Sorry, no magic bullet.

of course transformation efficiency decreases with plasmid size but as you get only the original vector I would rather suspect that something is wrong with your Gibson assembly. check the sequences of your primers. 20kb is not that huge size for plasmids...

I agree with Didier Poncet that 20kb should be ok and the difference in efficiency between 14kb and 20kb won't be that much. So look elsewhere for the problem.

Thanks for the responses.

Anatoliy Zubritskiy, I will try electroporation. What is the purpose of using lower growth temperatures?

Didier Poncet and Michael J. Benedik I will double check my Gibson Assembly design. However, My colony PCR was positive using one vector-specific and one insert-specific primer, so it seems that atleast one end of the Gibson Assembly was successful. Maybe I will use 2 flanking vector-specific primers to verify the assembly next time.

Aalap Parikh It's rooted in older times protocols for plasmids I can call big (around 100kbps or so), allows more time for replication.

The upper size limit for plasmid DNA that can be efficiently transformed into chemically competent E. coli TOP10 cells typically ranges between 10–15 kb under standard transformation conditions.

However, this efficiency drops significantly as plasmid size increases due to the physical and biological constraints of uptake. For plasmids larger than 15 kb, transformation efficiency decreases drastically, and alternative methods like electroporation or specialized strains engineered for large plasmids are recommended.

If you need to work with larger plasmids, consider using electroporation, as it can accommodate plasmids up to 300 kb or more, though the efficiency still decreases with size.

There is good correlation between the size of plasmid and transformation. There will be prompt decrease in transformation efficiency according to the size of plasmid

The size limit of plasmids that can be smoothly transformed into chemically competent TOP10 cells is generally around 15-20 kb, depending on the transformation protocol and cell quality. While ThermoFisher indicates that 20 kb plasmids should work, larger constructs like your 20 kb plasmid can pose challenges during heat shock transformations due to reduced uptake efficiency. Electroporation, which can handle larger plasmids more effectively, may be a better option for constructs of this size. Alternatively, you could consider using a strain specifically optimized for large plasmid transformation, such as E. coli strains like DH10B or XL10-Gold. Your colony PCR results may indeed reflect the successful assembly spread on the plate rather than actual plasmid uptake, as indicated by the sequencing and gel analysis. Optimizing transformation conditions, using fresh competent cells, or switching to electroporation should improve your chances of successfully transforming your construct.

Dear Aalap Parikh, I agree with Michael J. Benedik and Didier Poncet that the 20kb plasmid size should not be an issue. For a prompt solution, I suggest you try electroporation, which has a higher transformation efficiency than heat shock. For instance, electroporation transformation efficiencies may be as high as 2.0 x 1010 CFU/µg DNA, while heat shock transformation efficiencies are usually between 1.0 x 105 – 2.0 x 109 CFU/µg DNA.

The upper limit of plasmid size that can be efficiently transformed into chemically competent TOP10 E. coli cells is generally around 10–15 kb. This limit arises because larger plasmids are more difficult to take up through the cell membrane during the heat shock transformation process. Efficiency decreases significantly for plasmids larger than this range.

For plasmids larger than 15 kb, you might consider alternatives such as:

1. Electroporation: More efficient for larger DNA fragments (up to 50 kb or more).

2. Specialized strains: Use strains optimized for large plasmid transformation.

3. Linear DNA assembly systems: Such as in vitro recombination, which can bypass some size limitations.

Il n'y a pas de limite supérieure stricte pour la taille des plasmides pouvant être transformés dans les cellules TOP10 chimiquement compétentes. Cependant, l'efficacité de transformation diminue généralement avec l'augmentation de la taille du plasmide.

Voici quelques points à considérer :

• Efficacité de transformation: Les cellules TOP10 sont optimisées pour la transformation de plasmides de taille standard (jusqu'à environ 10 kb). Des plasmides plus grands peuvent être transformés, mais l'efficacité de transformation (nombre de colonies transformantes par microgramme d'ADN) sera probablement plus faible.
• Stabilité du plasmide: Les plasmides très grands peuvent être instables dans les cellules bactériennes, ce qui peut entraîner une perte du plasmide ou des réarrangements.
• Protocole de transformation: Il est important d'utiliser un protocole de transformation optimisé pour les cellules TOP10 et pour la taille du plasmide que vous utilisez.

Recommandations:

• Pour les plasmides de grande taille (plus de 10 kb), il est conseillé d'utiliser des cellules compétentes spécialement conçues pour la transformation de grands plasmides, ou d'utiliser des méthodes alternatives de transformation telles que l'électroporation.
• Il est toujours important de vérifier l'efficacité de transformation en réalisant des transformations témoins avec un plasmide de taille connue.

En résumé: Bien qu'il n'y ait pas de limite supérieure absolue, l'efficacité de transformation diminue avec l'augmentation de la taille du plasmide. Pour les plasmides de grande taille, il est recommandé d'utiliser des cellules compétentes ou des méthodes de transformation optimisées.

Contrary to a few of the above replies, size absolutely makes a large impact on transformation efficiency, particularly on non-supercoiled plasmids (such as Gibson reactions). This can easily be an order or two decrease relative to a small 3kb plasmid. Restreak several dozen colonies on a new plate to eliminate the potential Gibson reaction contamination before assaying by PCR. Check to see if the reaction can be cleaved with a restriction enzyme to eliminate non-insert containing molecules before transformation.

Thanks all for the helpful responses. I reviewed the primer design and indeed found some issues regarding secondary structure, so I will redesign my primers and reattempt with heat shock before trying electroporation. I'll update this thread with my results.

Yeah right, heat shock treatment before elecoporation technique and redesigning of primer will prompt accurate results

The upper limit on plasmid size that can be transformed into chemically competent TOP10 cells is generally around 10–15 kb. Efficiency decreases significantly as the plasmid size increases, with plasmids larger than 15 kb being challenging to transform via chemical methods.

Hi all. I was able to successfully complete the cloning after redesigning my primers such that the homology arms were extended to a length of 35 nt. I guess using longer homology arms to accomodate the relatively large insert (6 kb) was the key. I was able to successfully transform the ~20 kb product into TOP10 by heat shock, but growth of colonies and especially overnight cultures is noticeably slower with the larger plasmid.

Thanks for all the help!