it may help u

Saccharomyces cerevisiae strains were used:

DTY3  The yeast vector p424-

QsMT was constructed as follows: the QsMT coding region was

excised from pGEX-QsMT by digestion with BamHI/PstI and ligated

into the yeast expression vector p424 (Mumberg et al., 1995) under

the transcriptional control of the yeast GPD (glyceraldehyde-

3-phosphate dehydrogenase) promoter. The p424 vector also contains

the CYC1 (cytochrome c oxidase) terminator, the 2 l replication

origin and the TRP1 tryptophan marker. Vector p424 and the

construct p424-QsMT were introduced into cup1D cells using the

lithium acetate procedure (Stearns et al., 1991) and transformed cells

were selected by their capacity to grow in complete synthetic medium

(SC), lacking Trp and Ura (SC–Trp–Ura).

Thanks Kamlesh Shah, but it is this that I am doing. I am working with p424 and the problem is that I have colonies in the negative control, when I use SC-Trp-Ura.

I checked if the stock of DTY4 that I have could be wrong, but it is ok. I changed reactants, medium, pipettes and the problem remains.

I use the LiAc protocol to transform cells.

What medium are you using for selecting transformants, and how did you check the trp genotype of DTY4?

Hola Anna (sóc Mar)

Sometimes rich media contains aa enough for letting cells to grow even without extra addition. Just try to prepare selective plates with minimum media (SD instead YPD)

Dear Anna,

I think that two major issues can be involved. DTY4 is a W303 derived strain and for this reason it has the trp1-1 mutant allele. The allele contains a single mutation, so it can revert, although-I speak with experience-reversion is rare. But since you plate, after transformation, approximately 10(8) cells (I imagine) it is normal to find in the negative control 1-10 colonies (rarely more then 10, but if reversion occurs in the very first phase of growth you can have more than 100 colonies). Having said that, if the tansformation is well performed, according to the Gietz protocol, the transformation efficiency is approximately10(5) colonies per 10(8) total cells per 1 ug of plasmid. So if you transform with 100-200 ng of plasmid you should obtain on the plate 10(4) colonies, that is a number that, in my hands, is near to reality (as a matter of fact we plate normally 1/10 of the final transformation and obtain more than 1000 colonies). So there can be two I issues as I said: too few tranformants in your plate or two many revertants in you negative control. Let's say that if you obtain less than 200-300 colonies in your transformants plate, the problem is a too lower transformation efficiency. On the contrary, if there are more than 200-300 colonies in your negative control the problem is too much reversion. To reduce the probability of trp1-1 reversion, I suggest you to plate the DTY4 strain on medium with Trp in order to obtain single colonies. When the colonies are sufficiently big (but not to much) inoculate two of them in two independent cultures, and transform both of them, hoping that at least one is poorly reverted.

Best,

Enrico

Dear Anna:

 As explained perfectly by Enrico, trp1-1 mutation can revert. Therefore you should isolate several colonies from your strain and choose those maintaining Trp auxotrophy; then you can use any of those for transforming your pRS424 plasmids. The reversion frequency is low enough to be a problem using plasmids. However, if you have not been using single colonies you could have accumulate Trp+ suppressor in your strain, explaining the high rate of revertants.

We are using routinely W303 strains and TRP1 plasmid without any problem.

Cesar

Just plate the yeast cells on the -trp plates without transforming them to check whether they grow on it or not. This can tell you if there is a problem with the yeast cells or transformation experiment.

Hi Anna, I think that Enrico already gave you the best suggestions you could have. I might only stress that you start from one, or a few, tested and true trp- colonies for your pretransformation cultures(s). It may help to include extra tryptophan in the precultures, as it's frequently done with adenine, to countereselect possible TRP+ revertants. Even in my experience, however, reversion of the trp1-1 mutation appears extremely rare. Good luck

Hi everyone,

thanks for all your tips.

I try to answer all your comments:

Alejandro Martin: thanks Alejandro. Before doing the transformation, I plate DTY4 in SC-Ura plates and I pick only one colony to start the pre culture and do the transformation. Once the transformation is finished I plate the cells in SC-Ura-Trp medium (only the transformants should grow in that medium).

Mar martinez pastor: moltes gràcies guapa! I try to work always with SC-Ura medium to be sure that only DTY4 can grow there. Besitos a tots!! :*

Enrico Baruffini: thanks a lot Enrico. I try to follow your tips and I tell you if it works.

Cesar Roncero: thanks for your tips too. I follow also your tips.

Saira Dar: thanks. I did this previously and before the transformation, no colonies are in SC-Ura-Trp plates. Then, I supose the problem is during or after transformation.

Alessandro Fiori: thanks also. I will follow Enrico and Cesar tips and I tell you what happens.

Then... I go to work on this! :) :)

Hi to everyone,

as I promised to tell you "the end of the story" I send you this message.

After some weeks of work with DTY4, we ordered a "new" DTY4 from a collaborator group, but the problems with this new strain continued in the same way (maybe the LiAc protocol produces some change in the yeast).

We checked literature and we decide to repeat the transformation with the strain 51.2c (which has also the same auxotrophy and acts like DTY4, grows in SC-Ura-Trp when you put the p424 plasmid).

With only the first transformation I had good results: colonies in the plates with 51.2c + p424 and any in the negative control.

I do not know what could happen in DTY4 but finally the work with 51.2c allows me to end the story very well.

Thanks to all!!