Transformation of yeast - (Feb/14/2005 )

I wonder if someone can send me in the right direction, I am suppose to make a yeast transformation.

We have a strain of yeast that lacks the Ade1 gene and one that does have it in its genome. The task at hand is to transform the Ade1 gene into a vector (pAL19) and then send it into the strain that is missing the gene.

Here is how I have set up the steps:

1. DNA extraction from the wildtype

2. Running a PCR with the primer I have made and amplifying

3. Running the DNA in Electrophoresis

4. Cutting the DNA with the selected restriction enzyme

5. Introducing the fragment containing ade1 into the vector, the vector is opened with the same enzyme.

6. Introducing the vector to the ade1 lacking yeast.


Am I missing something? I might not have used the right phrases in english, but my other problem is finding lab protocols for all this, please help a noob :-(

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You have the basic concept right, but there seems to be some things missing. Your're vague on specifics so its hard to tell if you know what you need to be doing. The way I do it would be something like this:

1 - Get yeast genomic DNA somehow from the wt and clone ADE1 via PCR

2 - Run a small amount of the PCR product on a gel to verify the PCR was successful

3 - Purify the rest of the PCR product

4 - Digest and ligate the product into pAL19

5 - Make sure the ligation product is correct (free of mutations, etc) via sequencing.

6 - Transform the plasmid into your ade1 strain via the LiOAc method

7 - Select for transformants by plating onto media lacking leucine

8 - Pick a few colonies, grow them up seperately in media lacking leucine

9 - Test them for the prescence of ADE1 somehow (phenotypic evaluation or PCR genotyping) ade1 colonies should be red in color on media lacking adenine so you can compare your potential transformants with an ade control by streaking them on seperate plates, each lacking adenine. Dont forget to keep your potential transformants in/on media lacking leucine or they will lose the plasmid.

Good luck and reply back with any questions

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Thanks alot, now the questions that pop up are:

Step nr 3 is it really necessary? Because from what I have been told I am suppose to retrieve the gene directly from the gel with some form of a kit.

I am with you on all the other steps, it seems as if I was very very vague yesterday when I wrote this.

I can add that the restriction enzyme I have choosen for bothe the digestion of the yeast DNA and the vector is HindIII which will simplify the ligation of the ade1 gene with the pAL19 vector.


I have another question regarding the primer I have made with webprimer.

Now when I designed the primer I used ONLY the ade1 genes 921 baspairs! Did I do it right? Since I am doing a PCR and I only want those baspairs amplified. Should the primer start further up and downstream from the ade1 gene?

Once again thanks!

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Well yes you need to purify your product, but the method of purification can vary. When you run the whole PCR on a gel and cut out the band, you are purifying it (gel purification) because you are seperating your product from other stuff, then getting rid of the agarose.

The size of your PCR product would dictate the method of purification. Sounds like you know which method is best for your product, so your probably good.

I used to do alot of PCRs to do genotyping on the yeast knockout mutants and those products were ~500 bp. I ended up using the Geneclean 2 kit from Bio101, and the Qiagen PCR clean up kit.

One thing to remember - when you run any DNA on a gel, you are probably going to stain with EtBr and visualize your product with a UV light. This is sometimes not the best way to treat your valuable DNA, especially if you are going to do things to it later (i.e. ligation) The kits I used worked for me because of my PCR product length and I never had to gel purify them.

You should decide which method of purification is best after you determine your PCR product length. Typically one uses agarose gel purification for larger products and those quick spin kits for smaller products. The kits will tell you their limitations in their manuals, which you can normally get online in PDF format

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You might try checking out this webpage, as designing primers can be tricky business. This site is one of the best to learn about PCR and they have links to primer design strategies.

http://www.highveld.com/pages/pcr.html

Exactly how much homology you need to ADE1 or if you should include a region upstream and downdtream to ADE1, I cannot say as it depends on many factors . You might want to design your primers such that you can use one or both for sequencing primers as well.


There have been numerous posts on this forum regarding the topic though im sure

Another forum similar to this one might help you out as well

http://micro.nwfsc.noaa.gov/forums/index.php

Also, for anything dealing with yeast, Id refer to SGD:

http://www.yeastgenome.org/

type in ade1 in the search, then use the tools on the right to get the data you need

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