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Yeast high-efficiency transformation


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#1 orchidsbloom

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Posted 01 February 2014 - 07:39 AM

Hello everyone,

 

I had a really long day in lab and ended up spending the night here. Among the several things I had to do, I did a yeast high-efficiency transformation using lithium acetate. I exactly followed the protocol, added all the reagents in the order specified, heat shocked for 40 min at 42 degrees, took them out and centrifuged them. Something else came my way and I completely forgot about the cells and fell asleep for three hours. After I woke up, I realised that my cell pellet was sitting for three hours with the transformation mix as the supernatant part. Is my transformation useless now? Please help... I really really would like to know. I did plate the cells anyways but even if i get colonies, I am not sure I should trust them now! :( Please help!



#2 pito

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Posted 02 February 2014 - 02:03 AM

its possible they are just fine...(but hard to predict of course)

ANd why would you trow it out if they grow? Dont you use a selection , so that only transformed cells survive?

 

Just check the colonies you see....

No reason to just trow it out.

 

 


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 orchidsbloom

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Posted 02 February 2014 - 01:06 PM

Thanks for your reply. Yes, I do plate my transformants on selective media. But still I am being a bit paranoid because LiAc is a mutagen and having my cells (even though they were centrifuged and pelleted) exposed to LiAc for long might induce mutations in my strain background .. Am I thinking too much here or is it right to be this paranoid and treat my transformants with a grain of salt?



#4 pito

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Posted 02 February 2014 - 01:23 PM

Lithium acetate makes holes in the celwall, not sure its a mutagen, where did you get this information?

 

 

And yes: you might have some problems with your cells, but it does not mean everything is lost. It also depends on what you are doing and want to do with your cells.


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.





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