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

yes good idea

i will take a loop, scrape off the little dots, spread on normal LB plate and Strep LB plate. 

I will do the same (with another loop), scrape off the withish streaks on the wild type side and spread them on new normal LB and strep plates.

Then incubate and post the results.

But can't do this until after the 28th. In the mean time, my plate is in the fridge at 4C. Will enough bacterias survive until then ?

On Friday, 23 December 2016 15:13:44 UTC+1, Simon Field wrote:

This hypothesis should not be too difficult to test.

Perhaps viewing the non-dot areas under a microscope (perhaps with a stain for that species) would quickly find individuals, or taking a small sample of that area and streaking another plate to see if colonies form.

Cheaper than sequencing, and faster.

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On Thu, Dec 22, 2016 at 3:47 PM, Bryan Jones <bryan...@gmail.com> wrote:

It definitely looks like you have growth in both sides of the plate. The bottom doesn't have dots because the growth is too thick to see gaps between the colonies.

On Thu, Dec 22, 2016, 5:38 PM ukitel <marco.r...@gmail.com> wrote:

I'm afraid there is no streptomycin on the plates.
Can you describe in detail how you made the plates?

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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