Hello,

I use PCR cassette for yeast transformation. This PCR cassette-based transformation has much lower efficiency than the canonical integrative plasmid or non-integrative plasmid-based transformation. However, it is a better choice for a long gene or unknown gene. I am sharing some of my insights regarding this method.

1. I use Q5 hot start polymerase for the cassette amplification. Other high fidelity DNA polymerase will do fine job.

2. I use 45-55 nucleotide homology for designing of cassette primers. Often long primers contain secondary structure or form primer-dimer (predicted from OligoEvaluator). I use 1.1 M of betaine in the PCR mixture to lower the Tm and eliminate secondary structure-related issues.

3. I use commercial kit for the cleaning (purification) and concentrating PCR cassettes. I elute PCR cassette DNA with water instead of elution buffer (which probably contains Tris buffer and other salt). Water elution is better for cassette transformation.

4. I load approximately 1-2 microgram of PCR cassette DNA for transformation. PCR-cassette-based transformation has low yields, even in wildtype strain.

5. Before the transformation, I heat denature the singled stranded DNA at 95C for 2 min followed by cooling it down before adding it to the transformation mixture. I also prepare fresh dilution of lithium acetate solution each time.

6. After adding transformation mixture and mix it thoroughly, I use 30 min incubation at 28C in a shaker followed by heat shock treatment at 42C for 20 min in a water bath.

7. I use colony PCR for the verification of the recombination yield. No need to perform genomic DNA extraction. Pick a colony, resuspend it in 5 micro litre water, heat denature the colony at 95C for 5 min, then add 25 micro litre PCR mixture (we use low fidelity DreamTaq polymerase for this purpose). This is enough to verify the desired product.

Best wishes!

Satyendra Mondal Thank you for the advice, I really appreciate it!

I do not clean the amplified cassette before transformation. I usually amplify the DNA and confirm the size of the cassette is accurate through gel electrophoresis, then do phenyl chloroform and Ethanol Precipitation to have concentrate DNA for transformation after the confirmation. I resuspend my DNA in TE so perhaps that could cause issues with integration of my cassette into the accurate genome locus? Maybe I will try the PCR cleanup and cassette DNA water resuspension.

I have had issues with colony PCR for yeast as I find it inconsistent, sometimes working and sometimes not, so I decide to do a genomic DNA extraction. How is your efficiency? How much colonies do you screen on average until you find a successful integration with these methods?

Hello,

TE buffer may work fine because commercial elution buffers contain Tris, but EDTA may cause issues during transformation. I am not completely sure about it but you can try water elution as a comparison to your method.

Colony PCR is truly inconsistent and sometimes specific transformation requires standardisation of the protocol. I use step-up PCR where I start the PCR with an annealing temperature 5C lower than the suggested annealing temperature, increase the annealing temperature 0.5C in each cycle for 20 cycles (so after 20 cycles, the annealing temperature = starting annealing temperature + 10 C, or, suggested annealing temperature + 5C), followed by running the PCR with the suggested annealing temperature for 10 cycle.

To prepare the template DNA for the colony PCR, I pick up a small amount of colony and resuspend in 5 micro litre of water (if dilution is required, then dilute 10 times), denature the colony at 95C for 10 min before adding the PCR mastermix.

Also it depends the type of transformation and the sequence variance at the C-terminal (3' of the gene). The gene deletion is tricky sometimes. For fluorescent protein labelling, I check the colony first under fluorescent microscope and use those shiny ones for colony verification.

Best wishes!