Citing Richard Gomer <[log in to unmask]>:

> Someone told me, and we've seen, that to prevent mutations of the  
> Dicty AT-rich DNA in bacteria, you should do everything possible to  
> prevent stressing the bacteria:
> 1) grow at 30 C rather than 37 C
> 2) when growing the transformed bacteria on plates for bacterial  
> colonies, pull the plates when the colonies are pinpoint size, don't  
> let the colonies get bigger
> 3) when growing cells in liquid, use 1 ml of broth in a 1" diameter  
> test tube, have the tube slanted to maximize surface area exposed to  
> the air, and pull the culture when the density is 'heavy smoky',  
> don't let the culture go completely opaque
>
> cheers
>
> Richard Gomer
>
> ________________________________________
> From: DICTY [[log in to unmask]] on behalf of Chubb,  
> Jonathan [[log in to unmask]]
> Sent: Tuesday, May 21, 2019 10:28 AM
> To: [log in to unmask]
> Subject: Re: [DICTY] act15 promoter alternative
>
> Dear Mike,
>
> Many the promoters will show mutations every time you retransform  
> them into bacteria.  The long runs of As and Ts are unstable, even  
> without doing a PCR.  If the sequence matters to you, then you  
> should check at every cloning step.  In yeast, for TATA box genes,  
> small deletions and expansions do not seem to matter if the slippage  
> is upstream of the TATA, but downstream (between the TATA and the  
> TSS) it can be catastrophic.  I'm not surprised you see these  
> effects- certainly our A15 vectors have in the past got worse with  
> repetitive retransformation into bacteria.  If you look at the core  
> promoter-TSS region, it might provide some clues:
>
>
> GGATTCAAAAATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCAGATTGCATAAAAAGATTTTTTTTTTTTTTTTTTTCTTATTTCTTAAAACAAATAAATTAAATTAAATAAAAAATAAAAatg
>
>
> Jonathan
>
>



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