Re: [DIYbio] Re: My first successful CRISPR experiment on E. Coli ?

so as mentioned in my last post i streaked the "mutant" and the wt onto another plate with strep,

this time i took a very small qty of each colony and spread it as much as i could,

As you can see in below pictures (0, +6h, +9h and +30h after streaking), initially i could barely see any trace, then both colonies grew, maybe a little less on the wt side, but i don't know if it is significative.

So i would think i don't really have strep on this plate. 

I guess i would have to sequence both colonies to be really sure if i managed to edit the "mutant" or not.

On Thursday, 22 December 2016 20:20:47 UTC+1, Hugues wrote:

Hi guys,

i think i have successfully CRISPRed an E. Coli bacteria,

I have used the kit sold here:

http://www.the-odin.com/diy-bacterial-gene-engineering-crispr-kit/

I followed the protocol here:

https://docs.google.com/document/d/19RNlo-psr_kTeJtKOZK6ruFW18Uu7SIX4zZtHSc9dzQ/edit

The gene that was targeted is  Escherichia coli 30S ribosomal subunit protein S12 (rpsL) gene. One base pair was changed to render the bacteria resistant to streptomycin.

The picture attached shows my result. That plate contains a gel with streptomycin. The top part shows growth on the CRISPRed strain, the bottom part shows not growth on the normal strain.

What you guys think ?

I'm thinking of having the DNA of that gene sequenced, just to check if the edit really happened only where expected.

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