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

If you are not sure of streptavidin, you can add sterile strepatvidin solution on the top of the plates(0.5 ml - spread by tilting the plate), let dry.

Use two separate plates, one for WT, one for mutant. On each plate streak a loopful (about 10=20ul of bacteria) using the T-method (draw a T on the back of your dish, first streak the bacteria at the top of a T (red in diagram), then sterilize the loop, and pull down a streak from the top of the T down the leg of the T (yellow in diagram), sterilize loop again, and then starting from the bottom zigzag up (green in diagram) leaving about 1 to 2 cm from the cross line of the T (a sterile zone)). Incubate, you should get single colonies.

On Sun, Jan 15, 2017 at 8:55 AM, Hugues <lalibe...@gmail.com> wrote:

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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