Re: [DIYbio] Re: Any yeast culturing and/or gen transfer protocol, simple enough to do at a simple laboratory and sourcing yeast?

Don't know, sorry! :)

On 25/06/12 18:46, Andreas Sturm wrote:
> Lithium is no problem. have a look at
>
> http://www.youtube.com/watch?v=BliWUHSOalU
>
> Be sure not to hurt yourself by doing so.
>
>
>
> ( Sorry for OT, @Cathal, do you think, PEG works also with agrobacterium? )
>
>
>
> 2012/6/24 shamrock <thmsburkett@gmail.com>
>
>> I'll have to try the Lithium free transformation, sounds interesting.
>>
>> On Sunday, June 24, 2012 3:56:03 PM UTC-4, Cathal wrote:
>>
>>> Ages back on the list we discussed Yeast transformation without lithium,
>>> because lithium's hard to get (at least over here), probably because of its
>>> medicinal uses.
>>>
>>> Turns out you can do a *really* inefficient yeast transformation with PEG
>>> 3350 (available as a laxative, brandnames "GoLytely" and "Miralax" should
>>> be easy to find online) and table salt. I'll dig up the protocol when I get
>>> a chance, but it ought to be in the list archives, too.
>>>
>>> If you have access to an enzyme that digests Yeast cell walls, you can do
>>> more high-efficiency transformations; I have a plasmid that produces an
>>> enzyme called "Lyticase" that does just that. I've never tested it..
>>>
>>> On 24 June 2012 19:58, shamrock <thmsburkett@gmail.com> wrote:
>>>
>>>> Hi Brian, ******
>>>>
>>>> Bakers yeast (Saccharomyces cerevisiae) is just as easy to work with as
>>>> E. coli, keeping in mind a few things. One minor complication is that they
>>>> grow slower and most antibiotics don't work as selections. What that means
>>>> from a practical standpoint is that contamination becomes a slightly
>>>> greater problem then when working with E. coli, and the media is a bit more
>>>> complex to make. However if you keep a clean environment and use good
>>>> aseptic techniques you shouldn't have much of a problem. ****
>>>>
>>>>
>>>>
>>>> The background yeast strain used for most genetic experiments is one
>>>> known as S288C. Other strains and backgrounds are used for a variety of
>>>> industrial, brewing and baking purposes but S288C is the one most often
>>>> used in genetic experiments. It has the advantage that it does not have a
>>>> lot of chromosomal duplications and gene amplifications that are found in
>>>> most industrial strains, plus a wide variety of different strain types are
>>>> avaiilable.
>>>>
>>>>
>>>>
>>>> There are a number of repositories that have collections of yeast
>>>> strains-alas I do not know how willing they would be to send something to a
>>>> home biologist. You could search on the internet for biological
>>>> repositories or if you have access the latest issue of journal of
>>>> industrial microbiology and biotechnology has a review on yeast culture
>>>> collections (Boundy-Mills, K. 2012. Yeast culture collections of the world:
>>>> meeting the needs of industrial researchers. JIMB 39:673). ****
>>>>
>>>>
>>>>
>>>> Like I said earlier working with yeast is just as easy as E. coli with a
>>>> few caveats. First of all because most antibiotics don't work the most
>>>> often used selections are nutritional selections. What this means is that
>>>> the starting strain is unable to make a particular nutrient (usually an
>>>> amino acid or a nucleotide precursor) because of a mutation in a single
>>>> gene. The selectable marker carried on the plasmid has an unmutated copy of
>>>> the same gene. Therefore cells carrying the plasmid can grow in the absence
>>>> of the nutrient but cells that do not have the plasmid cannot-just like E.
>>>> coli cells with a plasmid can grow in the presence of ampicillin but cells
>>>> that don't have the plasmid cannot. From a practical standpoint it means
>>>> that the media used is a bit more complex so instead of growing cells in
>>>> rich media (which is relatively simple) you need to select for recombinant
>>>> cells on media that lacks only that single nutrient. You can but the media
>>>> known as drop-out media or you can make it yourself but it is more complex
>>>> to make from scratch. Cells are transformed similar to E. coli cells
>>>> although the method used to make the cells competent to take up DNA is
>>>> different. With yeast Li salts (Li acetate for cerevisiae and LiCl for
>>>> Pichia ) are used. You can also electroporate yeast like you can E. coli,
>>>> and there is a method based on digesting away the outer cell wall. ****
>>>>
>>>> A number of different plasmid vectors exist for use in yeast and a
>>>> number of different inducible promoters are available as well. So you can
>>>> make use of high oor low copy episomal vectors or you can use integrating
>>>> vectors. Promoters, such as the GAL promoter, can be turned on just by
>>>> shifting the carbon source from glucose to galactose (making sure the
>>>> glucose is used up). One really cool thing about yeast is that it is
>>>> relatively easy to make genome modifications either by knocking out certain
>>>> genes or adding new ones. Plus you can do some nice genetic experiments
>>>> through simple mating and sporulation. ****
>>>>
>>>> Working with yeast is fun, pretty easy to do, and they smell a lot
>>>> better than E. coli! ****
>>>>
>>>> On Saturday, June 23, 2012 4:15:23 AM UTC-4, Brian Soe wrote:
>>>>
>>>>> Hi friends
>>>>>
>>>>> I want to learn some biology experiment at home for fun. I wonder
>>>>> is there any yeast culturing protocol that can be done without a
>>>>> research grade laboratory but a simple bench, a light microscope,
>>>>> micro pipette and syringe, etc. May I know where are the potential
>>>>> sources where can I get them? Thanks.
>>>>>
>>>>> Sincerely
>>>>>
>>>>> Aung
>>>>>
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>>>
>>>
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