I am not an expert in this field, but I am very interested and have researched to find an answer. I received some assistance from tlooto.com for this response. Could you please review the response below to see if it is correct?

Proceeding directly to yeast cell transformation without purifying the DNA after Golden Gate assembly could be feasible but carries certain risks. Residual enzymes and reaction components from the assembly mix may inhibit transformation efficiency or introduce variability [2]. However, some studies have shown that transformation can occur under non-artificial conditions or by using simplified protocols [1][4]. Methods such as heat inactivation or commercial enzyme deactivation reagents can neutralize enzymes, while dilution can reduce inhibitory component concentration [3]. Although these methods might lower transformation efficiency compared to purified DNA, running test reactions to optimize conditions is advisable.

Reference

[1] Agmon, N., Mitchell, L., Cai, Y., Cai, Y., Ikushima, S., Chuang, J., Zheng, A., Choi, W. J., Martin, J. A., Caravelli, K., Stracquadanio, G., & Boeke, J. (2015). Yeast Golden Gate (yGG) for the Efficient Assembly of S. cerevisiae Transcription Units.. ACS synthetic biology, 4 7, 853-9 .

[2] Mizukami, A., Nagamori, E., Takakura, Y., Matsunaga, S., Kaneko, Y., Kajiyama, S., Harashima, S., Kobayashi, A., & Fukui, K. (2003). Transformation of yeast using calcium alginate microbeads with surface-immobilized chromosomal DNA.. BioTechniques, 35 4, 734-6, 738-40 .

[3] Nevoigt, E., Fassbender, A., & Stahl, U. (2000). Cells of the yeast Saccharomyces cerevisiae are transformable by DNA under non‐artificial conditions. Yeast, 16.

[4] Polaina, J., & Adam, A. C. (1991). A fast procedure for yeast DNA purification.. Nucleic acids research, 19 19, 5443 .

Hi Andrea, if you do not need a ligation step, you can simply heat up your plate to 75-80˚C for 10 minutes (this deactivates the ER) and continue with transformation. I assume the cassette you mentioned is a linear DNA, so it should work in my experience.

I don't think clean up in necessary for that transformation. However, QIAquick 96 PCR Purification kit does that—but the price is way too high.

Thanks to your answers!

Park Sowon, I reviewed the literature and didn't really found a solution for my question however, they are good articles and source of many information. Thanks for sharing.

Hamid, indeed after RE digestion I will end up with a mix of linear DNA fragments. The denaturation step is an option I considered too however, I'm more concerned about the presence of the buffer. The transformation efficiency of my strain is already not great, so I’d prefer not to reduce it any further. Do you have an opinion about it?

You can digest a few µg of DNA in 10-20µL and then bring the volume to 50µL. If your transformation method recommends at least 300-400µL of total transformation mixture, I think the dilution is significant, and you'll be fine. I would highly recommend a test run since it saves lots of time.

If you really need the cleanest DNA, then I recommend using a chip PCR cleanup kit.

Btw, can you not PCR amplify that linear PCR instead of cutting it with ER? That would give you a big amount of DNA.

PCR amplification of the cassette is not recommended because the single units contain functional genes and are connected by small homologous regions. Any SNP introduced by the polymerase could be disastrous therefore, additional sequencing would be required to confirm the sequence, which will be both expensive and time-consuming.

Diluting the samples is definitely a good option, but excessive dilution will also reduce the transformation efficiency. I think I will try to compare different transformation playing with DNA amount, RE enzymes deactivation and dilution.

Thanks