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Cloning

Genetics as it applies to evolution, molecular biology, and medical aspects.

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Cloning

Postby Agronomy » Fri Feb 23, 2007 6:34 am

We did this lab in one of our labs, where we transformed our ligation reaction (TOPO vector + TTG PCR product) into E.Coli competent cells and plated them onto agar plates (LB + kanamycin + Xgal).

Supposed that the next day we inoculated one blue and one white colony into separate test tubes containing 3ml of liquid media (LB and kanamycin) and allowed them to grow overnight.

After the cells grow, we realize that we didn't label our tubes, and now we have to determine which tube contains the recombinant plasmid before the end of the day.

The only enzyme available is SacI.

How can we tell how we would identify which tube contains the recombinant plasmid?

I am TOTALLY lost on how to answer this...
Any help would be appreciated, thank you :-)
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Postby Vagabond » Fri Feb 23, 2007 2:42 pm

I am sure there are a number of ways to do this but off the top of my head here. Do you need to use the SacI enz? If so, do a small mini prep of each sample digest it and run a gel done. Or just do a mini prep and run a gel the one with the plasmid should be obvious. Why would you pick a negative colony anyway and why would you not label them?
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Postby Agronomy » Fri Feb 23, 2007 5:52 pm

it's actually a question that our TA brought up in lab, like just a scenario...and we just have to like think about it...and ya we have to use that Sac enzyme.
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Postby canalon » Fri Feb 23, 2007 9:21 pm

I do not have a map of the TOP vector but I would extract the DNA of my suspension and digest it with Sac1 and compare the size of the restriction fragments.

I wonder, although I never tried if you could not just add some X-gal in the solution and incubate a few hours at 37ºC to see the culture turn blue/green...
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Postby Agronomy » Mon Feb 26, 2007 10:06 pm

if I did have a map of the TOP vector, which I do (in my lab manual) then what would I do with it?
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Postby Agronomy » Mon Feb 26, 2007 10:20 pm

I actually found one spot on the entire sequence with SacI and it says bp 1286...there's only one recoznition site with it though, what should I do with that?
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Postby Vagabond » Mon Feb 26, 2007 11:12 pm

if only one reatriction cut site then if you prep the plasmid and use the enzyme you will get a single linear band of your plasmid the other prep will not.
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Postby canalon » Tue Feb 27, 2007 4:17 am

Vagabond wrote:if only one reatriction cut site then if you prep the plasmid and use the enzyme you will get a single linear band of your plasmid the other prep will not.


And you can compare the size of the band with a molecular weight marker, you will then know if you have an insert or not.
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Postby Agronomy » Tue Feb 27, 2007 3:34 pm

thanx for all your help :-)
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