Mmagerko
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Postby Mmagerko » Sat Sep 17, 2016 10:01 pm

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Last edited by Mmagerko on Fri Dec 30, 2016 11:29 pm, edited 1 time in total.

donnahardy2
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Re: Counting E. Coli colonies in ODIN CRISPR Kit

Postby donnahardy2 » Sun Sep 18, 2016 6:38 pm

Hi Mmagerko,

Welcome to Science Buddies!

The E. coli colonies are going to be discrete round, opaque, whitish, shiny dots on the surface of the agar. Unfortunately, I cannot access the instructions for this kit, so I’m not sure of the details of the experiment, but typically antibiotics are included so that any colony that grows has been transformed. If you are cloning for colonies that contain green fluorescent protein, the colonies will be green.

I don’t recommend proceeding with this until we can check on the details. This kit is new to this website, so I will ask a moderator if we can get the specific instructions. I should then be able to answer all of your questions.
Please let me know if you need any additional information.

Donna Hardy

Mmagerko
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Postby Mmagerko » Sun Sep 18, 2016 8:34 pm

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Last edited by Mmagerko on Fri Dec 30, 2016 11:29 pm, edited 1 time in total.

donnahardy2
Former Expert
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Re: Counting E. Coli colonies in ODIN CRISPR Kit

Postby donnahardy2 » Mon Sep 19, 2016 2:10 pm

Hi Mmagerko,

Thanks for posting the instruction manual. That’s what I needed.

This kit includes a gene that provides streptomycin resistance to the E. coli. So, after you do the transformation step, the only colonies that will grow will be the ones that have acquired the streptomycin-resistance gene. The original E. coli will not grow on the streptomycin plates. The streptomycin-resistant have the same appearance as the original strain.

I hope this helps. Please let me know if you have any other questions.

Donna

Mmagerko
Posts: 14
Joined: Sat Sep 17, 2016 9:50 pm
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Postby Mmagerko » Mon Sep 19, 2016 7:25 pm

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Last edited by Mmagerko on Fri Dec 30, 2016 11:29 pm, edited 6 times in total.

Mmagerko
Posts: 14
Joined: Sat Sep 17, 2016 9:50 pm
Occupation: Student

Re: Counting E. Coli colonies in ODIN CRISPR Kit

Postby Mmagerko » Mon Sep 19, 2016 10:29 pm

In addition to counting colonies, I have another question.

The instructions to edit the E. Coli and grow the colonies are the streptomycin media are concise, but the other part in my experiment has to do with growing the E. Coli without editing it and without the streptomycin media. It is possible, correct? I mean wouldn't you just use the E. Coli strain, LB Agar, and the petri dish? But I'm a little confused on how the process works, as in how long to incubate or let dry the plates without the transformation part.

Would really appreciate your help AAE :)

donnahardy2
Former Expert
Posts: 2671
Joined: Mon Nov 14, 2005 12:45 pm

Re: Counting E. Coli colonies in ODIN CRISPR Kit

Postby donnahardy2 » Tue Sep 20, 2016 8:00 am

You are correct. The E. coli included in the kit has not been transformed and will grow on any bacterial medium, such as the LB agar without streptomycin. After it has been transformed, the E. coli will grow on the streptomycin agar and would also grow on LB agar without streptomycin.

Since the only difference between the original and the transformed E. coli is the streptomycin-resistance gene, there would be no physical difference in the appearance of the colonies, but only the E. coli that have been transformed will grow on the streptomycin agar.

In the first part of the experiment, you will be growing the original E. coli on LB agar. In the second part of the experiment, you will grow only transformed bacteria on the streptomycin agar.

Counting colonies. On the last page of the instruction manual, there is an agar plate with white colonies growing on it. The agar plate contains bacteria that have been transformed, so they are able to grow in the presence of streptomycin. Each organism that has been transformed will grow into a whitish visible circle on the agar called a colony. To count these, you just make a note of each white dot and add up the total number of white dots on the plate. This is the total plate or colony count. It looks like there’s about 30 colonies on the plate in the photograph.

It’s possible to visually count colonies on the plate when there about 30-300 colonies present. With more than 300/plate, the plate surface gets crowded and it’s difficult to get an accurate count. That why people sometime divide the plate into 4 sections with a marker and count just one fourth of the plate at a time. It's just to make it a little easier to get an accurate count.

I hope this helps. Let me know if there are other questions. You are smart to keep asking questions until the experiment makes sense. This is a complicated experiment, especially if you have never grown bacteria before.

Donna


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