Cloning

February 23, 2007 at 6:34 am #7010

Participant

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 🙂

February 23, 2007 at 2:42 pm #69378

Vagabond

Participant

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?

February 23, 2007 at 5:52 pm #69384

Agronomy

Participant

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.

February 23, 2007 at 9:21 pm #69397

canalon

Participant

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…

February 26, 2007 at 10:06 pm #69488

Agronomy

Participant

if I did have a map of the TOP vector, which I do (in my lab manual) then what would I do with it?

February 26, 2007 at 10:20 pm #69489

Agronomy

Participant

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?

February 26, 2007 at 11:12 pm #69492

Vagabond

Participant

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.

February 27, 2007 at 4:17 am #69500

canalon

Participant

quote Vagabond:

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.

February 27, 2007 at 3:34 pm #69513

Agronomy

Participant

thanx for all your help 🙂

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