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Ubriaco
10/31/06 08:43 AM  
question for Al B
Al,

Is there something I can put in my plates that will select for S.c. specifically?

Alternatively, could I possibly get a kan plasmid that is for E. coli into and expressing in my S.c. library, or is the codon usage too different? I have a ton of plasmids with kan resistance laying around.

thanks,

vinnie the drunk

Ubriaco
10/31/06 08:59 AM  
Re: question for Al B
Just thought of one thing: What if I adjusted the pH of my media down to about 4, that would do it wouldnt it?
Al B
10/31/06 09:52 AM  
Re: question for Al B
Ubriaco -

Question 1: Well, I know that metabisulfites will supress Bretts and bacteria, but the concentration is critical. Too much and the Sacch. will be inhibited. I will need to look further. Also, there are wild Saccharomyces (not cerevisae) that may have to be dealt with.

Question 2: Whaaaa?!?

Question 3: No, Lactobacillus can grow (small pinpoint colonies in the presence of air) Bretts will grow at pH of 4, no problem. But you could combine a low pH with something else, like sulfites.

Back to Question 2: Say Whaaaaaat?!?!?! (that one went waaay over my head.....and it hurts) sorry.

Ubriaco
10/31/06 10:30 AM  
Re: question for Al B
Good points, im growing on plates of the formula:

for 1L

3g/L malt extract

3g/L Bovine Casein

3g/L yeast extract

20g/L glucose

15g/L agar

I streaked the plates yesterday and to my surprise the colonies look much smaller than I expected, but they are all uniform, barely off-white, with only a minute amount of slimeyness. And they smell wonderful. Im pretty sure I dont have any contamination, as there is absolutely no variation in appearance of the colonies (correct me if that assumtion is incorrect). So I proceeded to the next step of selecting a few with the loop and putting them into broth for stepping up. After I get a saturated culture, I will be putting them into the -80 with glycerol.

I was just wondering if I could make sure that S.c. was the only thing growing, but it looks like that isnt absolutely necessary.

Question 2 came from me drinking heavily last night thinking about awesome ways to incorporate molecular genetic techniques into brewing. But since thinking about it more, I answered my own question -----an E.coli promoter and an Sacc. cerv. promoter are completely different, and a kanamycin resistance gene designed for one will not express in the other. Wishfull thinking, I could get one that would work in a eukaryote, but then Id have to explain what its for and I doubt that would fly.

Al B
10/31/06 10:41 AM  
Re: question for Al B
I usually recommend growing isolated colonies for more than 1-2 days to ensure purity (sometimes others grow more slowly) from a mixed culture. I then subculture again from an isolated colony to another plate. From there I prefer to use alot of cells when freezing with glycerol - to ensure viability (greater in numbers).

Put all your plasmids in a fly and see what happens! Happy Halloween.

Al Bacteriaphage

Ubriaco
10/31/06 11:20 AM  
Re: question for Al B
Thanks,

thats good advice that I will follow as soon as I get some time today.

Hmm, in a fly......could be interesting, Happy Halloween

 
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