About expanding pre-made genome-wide sgRNA library.

[Heonseok Kim]

Hi, I have question about expanding sgRNA libraries.

When I made any kind of library, I did it by having "single colonies" 100x to the complexity of library in 24cm square plates.

So I didn't pour electroporated e.coli too much because every colony will be mixed if there are too many colonies than the area of plate.

(eg, 100,000 colonies in 24cm plate is OK but 10,000,000 colonies in 24cm plate would be too much)

But, according to Gecko library expansion protocol, they recommend to make ~3,000,000 colonies in 24cm square plate.

I think with this number, most of the colony couldn't be "single" but "mixed".

Is this OK for containing the complexity of library?

I'm curious about your opinion.

Thanks

[Neville Sanjana]

Hi Heonseok,

You can (and should) sequence your library after the maxiprep to verify uniformity and near-full representation. Even when I cannot see individual colonies (i.e. looks more like a uniform lawn), I have found that the amplification still works without problems in representation. 

In terms of colony number, I'd recommend making sure you have at least 30x (colonies per library construct) but usually retransformation of an existing library should yield a much higher representation.

Hope that helps,

Neville

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[Heonseok Kim]

"Unoform lawn" is exactly what i get today. 

Now I can proceed with it.

Thank you very much Neville

Best wishes,

2016년 2월 24일 수요일 오전 10시 50분 56초 UTC+9, Neville Sanjana 님의 말: