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

thanks for all the feed-back guys, this is kind of a peer review of my first paper published on the DIY Biologist, LOL.

The next few days are a bit busy, but i'll come back next week with specific feed-back and potential extra test to validate my hypothesis, before i shed a couple hundred bucks on having the little critters sequenced.

I don't have all the consumables to run another experiment but I still have normal Agar plates and some with Strep, so if someone has an idea how i can use these plates to further validate (or falsify) my hypothesis then your proposals are mostly welcomed.

thanks

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.