Recapitulating what several others have said, 5-6 days is way too long for transformants to grow, even on synthetic plates (3-4 max to get colonies several mm in diameter). The question is whether the transformants are real or not. NEVER inoculate transformant colonies directly into liquid media. They rarely grow well. ALWAYS streak transformants out again on selective plates to check for growth and obtain pure clones.

If you streak out your transformants on SD-ura, and they don't grow, they were background colonies. If you resuspended the transformed cells in YPD instead of SD-ura/water before plating them out, you're likely to get some background growth of non-transformed colonies. Also, using a few ul of mini prepped episomal plasmid (2u or CEN) should yield 100 or more colonies from a standard lithium acetate transformation. If you only see a dozen or so colonies, you might want to check your mini preps or the strain again (as a previous commentator said, good transformations require exponentially growing cells). The few colonies you got could be background growth or your transformations might be inefficient. If you get a lawn of cells, your starting strain is probably not really ura3.

If you streak out to singles on SD-ura and they do grow, but still slowly, there is something wrong with the transformants. Streaking out on FOA plates to select against the plasmid will tell you if growth is affected by the plasmids or not. If cells grow better on FOA, your plasmids are causing the slow growth (I really don't see how pYES2 could do this unless it's not really empty). If transformants grow just as slow or slower on FOA as SD-ura, there's something wrong with your starting strain. Streaking out the starting strain on FOA at the same time will act as a growth control.

In my experience, healthy yeast colonies (such as those obtained transforming with pYES2) reach a good size (2-3 mm diameter) in 3 days at 30C after a transformation such as the one you made. The fact that they took 5 to 6 days suggests to me that there was something wrong in the transformation procedure and that the colonies you obtained are actually a contamination with non S.cerevisiae yeast. You could check the cells from your colonies in the microscope. Usually (not always), contaminating yeast are smaller than S.cerevisiae.

One control you should do that will test that your medium and everything else was in good shape, is to add uracil to the liquid medium you used and grow in it the parental, unstransformed strain you used in the transformation. Most common lab strains grow with a doubling time of 90 minutes approximately.

During your selection, after transformation, did you also plate a positive and negative control strain onto your -URA medium? After you transform and see colonies growing I would pick 10-20 colonies and then re-streak onto -URA medium. This will aid single colony isolation and confirm your phenotype. (1) You could have mixed genotypes. (2) you may have not integrated your marker in all colonies and they could be episomal carriers. (3) The marker may have integrated but in a spot that hampers growth/maintenance.

All colonies at this point should be maintained on -URA medium. Then I would do a colony PCR to confirm the integration of the URA marker. Using one primer located in the URA marker and another outside the expected integration site.

Slow growth of a strain after transformation is usually indicative that the insert is toxic to the cell, however, it is unlikely that this is the case here, as both the vector alone and vector + insert appear to grow slowly and I assume that you are plating the transformed cells on SC-ura containing 2% glucose, so the HSP104 gene should be 'off' (if it is under the pGAL promoter).

If the strains grow slowly both after transformation and when inoculated from a patch into liquid culture (so you have a 'good' amount of innoculum), I would suspect that there is a problem with the expression of the URA3 gene and suggest making the construct again. Adding uracil to the culture to see if the growth rate increases will give you an indication as to whether this is the problem before you clone the HSP104 insert again.

Is URA gene mutated in your strain matching with the one inside the vector? Normally is URA3...This a stupid question, but a starting point to understand your problem. 5-6 days are a bit too much for yeast URA selection, usually 2-3 days are enough. I'm thinking that the colonies that grew were not your yeast transformants, but contaminants.

In my experience, there might be two problems:

1.the yeasts are very old, i.e. are grown on the plate for more than two weeks before transformation. Old yeasts grow very slowly on SM media after transformation, especially when DMSO is used for transformaton.

2.the yeasts are obtained some spontaneous mutation due to high number of passages from plate to plate.

To solve, the first problem just grow fresh yeasts and use transformation protocol with preparing overnight culture.

To solve the second problem, the original yeast culture should be used.

I suggest to follow this protocol:

HIGH EFFICIENCY TRANSFORMATION OF SACCHAROMYCES CEREVISIAE BY ELECTROPORATION (Manivasakam, P. and Schiestl, R.H. (1993) NARS 21, 4414-4415)

High efficiency transformation of By4742 was carried out as follow:

1. Inoculate 5mL of YPAD medium and grow overnight at 29-30ºC. Inoculate cells into 100 mL of liquid YPAD medium and grow until 3.2 x 107 cells/mL (OD600= 1.3-1.5).

2. Harvest the cells and wash twice with 100 mL sterile water and resuspend in 200 µL sterile water. This is enough for 2 transformations.

3. Add 100µL cell suspension to 50 µL solution* containing 100-150 ng plasmid DNA and 20 µg of single stranded carrier DNA and mix thoroughly.

4. Add 250 µL 50% PEG 4000 and mix by brief vorting.

5. Incubate the mixture at 42ºC for 15 min.

6. Transfer this mixture to a 0.2 cm electroporation cuvette and electroporate at 1.5 kV, 25 µF and 200Ω (BioRad Gene Pulser with Pulse controller, program Sc2).

7. Immediately add 600 µL ice cold YPAD medium.

8. Plate the cells onto selective medium with appropriate dilution.

9. Incubate the plates for 3-4 days to obtain transformants (in your case Sc-ura).

* Solution used contained: 10µL ssDNA, 1µL pGREG575/SalI, 15µL and 30µL of PCR products, sterilized H20 up to 50µL of volume.

** By4742 and two solutions without PCR products (in your case, salmon sperma DNA + pYES2 and pYES2+Hsp104) were used as controls.

SELECTION AND GROWTH OF THE TRANSFORMANTS

Only the yeast cells that have incorporated the transforming DNA (yeast expression plasmid) are able to grow on plates lacking a specific amino acid. Positive transformants containing the pYES2 and pYES2+Hsp104 plasmid are selected on Dropout Medium (DM) minus uracil plates.

500 mL of DM medium contains 3.3 g of Yeast Nitrogen Base w/o aminoacids, 10g glucose, 0.41 g drop-out (all aa’ minus ura). At 28-30ºC during 2-3 days is enough.

Good Luck!

could be the type of sugar you are using as a carbon source in the media. I work with 2 different yeast strains BCY123 and wt303. The BCY123 (for us) average twice as long as the wt303 strains regardless off the media, which could be about 48hrs (BCY strain). We use 3 different sugars depending on what we are trying to accomplish with the gene of interest. We use glucose, raffinose or galactose in our minimal media. Raffinose and galactose both cause the yeast to grow a lot slower.

But I also agree with Alejandro: healthy yeast usually only takes about 2-3 days after transformation plating and 2 days to in liquid culture to have visual indication of growth.

Hi,

for some strains u will see this kind of growth pattern. what u can do is once u get transformants restreak them in a fresh selection plate. then u go for liquid culture.

This way long lag phase can be reduced by giving fresh media supplement.

Recapitulating what several others have said, 5-6 days is way too long for transformants to grow, even on synthetic plates (3-4 max to get colonies several mm in diameter). The question is whether the transformants are real or not. NEVER inoculate transformant colonies directly into liquid media. They rarely grow well. ALWAYS streak transformants out again on selective plates to check for growth and obtain pure clones.

If you streak out your transformants on SD-ura, and they don't grow, they were background colonies. If you resuspended the transformed cells in YPD instead of SD-ura/water before plating them out, you're likely to get some background growth of non-transformed colonies. Also, using a few ul of mini prepped episomal plasmid (2u or CEN) should yield 100 or more colonies from a standard lithium acetate transformation. If you only see a dozen or so colonies, you might want to check your mini preps or the strain again (as a previous commentator said, good transformations require exponentially growing cells). The few colonies you got could be background growth or your transformations might be inefficient. If you get a lawn of cells, your starting strain is probably not really ura3.

If you streak out to singles on SD-ura and they do grow, but still slowly, there is something wrong with the transformants. Streaking out on FOA plates to select against the plasmid will tell you if growth is affected by the plasmids or not. If cells grow better on FOA, your plasmids are causing the slow growth (I really don't see how pYES2 could do this unless it's not really empty). If transformants grow just as slow or slower on FOA as SD-ura, there's something wrong with your starting strain. Streaking out the starting strain on FOA at the same time will act as a growth control.

Thanks very much for the all comments. I'll try some and see what I get. Meanwhile further comments are welcomed.

These are all really good suggestions, I just wanted to add that we've worked extensively with overexpressing Hsp104 from a URA3-marked GAL1/10-promoter plasmid, and it is not at all toxic under any conditions to wild-type cells. Our cultures inoculated directly from colonies on a transformation plate grow just fine.

Yeast cells exhibits slow growth after so many generations of subculture. You are recommended to revive a fresh host culture from stock (glycerol) and check for growth rate. If it grows well, this can be used for your transformation..

I guess culture media of each company are not the same for you what is the best ?

Perhaps you can test another media ?

That is true. We tested several YNB media from various companies and at the end we found that the one we used was feigned, as the supplier confirmed he was not sure of its quality!!!, the other two also showed different growth rates. Thank you very much for the all answers. The problem eventually solved!

Can anyone troubleshoot my URA3- selection problem?

I am trying to make URA3 mutant of an yeast strain. I have prepared FOA plates and when I carry out transformation using salmon sperm dna with my vector containing insert, kanamycin gene flanking URA3 sequences (to delete the URA3 by homologous recombination) , I am getting colonies both in reaction plates and in control (where no vector has been added, only contains wild type strain). The colonies from ligation plates when they are grown in minimal media with uracil are able to grow, but without uracil, there is no growth on plates. How can I check whether the colonies are URA3 mutants or not? I even streaked the wild type on FOA plates, it showed colonies after 36 hours. Is there any biochemical way to test that FOA is working?

TOPICS

Since you are saying the you are getting background colonies in Kanamycin negative control plates you better check the quality of the selection plates first then proceed with next set of experiments.

FOA works efficiently only when excess of uracil is added to the media and replica plated second time.

sorry to bust in. but i can't find protocol for plates selective to PYES2, maybe you can help me?

it's spouse to be -ura right? so how do i make then?

and what is FOA for? i just lost it :)

i want to make sure my transformation did work and that my PYES2 is in the yeast before i continue

thanks

may

here is the recipe for -ura plate:

Glucose 2% w/v

Yeast nitrogen base with ammonium sulphate but without amino acids (YNB) 0.67% w/v

Agar (for solid media) 2% w/v

The amino acid drop-out mix (-URA from Formedium, UK) 0.2%

Here you don't need FOA, but you can grow your yest cells before pYes2 transformation (in case they have re-arranged their URA3 gene to a functional Ura3) in 5FOA to make sure they have lost Ura3 function.

5FOA is used for selecting strains which have lost a URA3 plasmid or gene. 5FOA is converted to the toxic 5 fluorouracil in the Ura+ cells.

how to make foa media? here is the recipe and protocol

YNB-5FOA [Yeast nitrogen base with ammonium sulphate but without amino acids 0.67% w/v; Glucose 2% w/v; appropriate amino acid supplements depending on which amino acid genes are mutated in the yeast strain; Agar 2% w/v]. Medium was then autoclaved at 121°C for 15 minutes and kept in a water bath 50ºC until required. In a separate bottle 100mg 5-FOA was added to 50 ml de-ionised water and heated to 50ºC with constant stirring until the 5FOA was fully dissolved. The 5FOA solution was then filter-sterilised and added to the medium which was kept at 50ºC, to make a final volume of 100 ml to give a final 5FOA concentration of 1mg/ml.

you can also extract your plasmid from yeast cells with commercial kits and sequence your cloned gene if you really need to make sure your gene sequence is correct, or simply you can do a colony pcr with a fresh colony. good luck.

thank you so much @behrooz moosavi :)

sorry to interrupt my question is can yeast wild type say strain W303 with no plasmid grow on selective media? Or it can only grow on YPD/A?