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