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Cloning Problems/ Help


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#1 pacemarshall

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Posted 31 October 2010 - 01:27 PM

Need help!

I am currently taking a Molecular Genetics course and I am having a little trouble with a problem.

Question:

Explain how you would clone a 1-kb ScaI fragment of nematode DNA into the PstI site of pBR322. How might you use selective media to identify and differnetiate non-transformed cells, cells with pBR322 alone, and cells with pBr322 and the nematode DNA insert?

I think I know how to identify transformed cells but I don't know how to answer the first part of this question. Any help would be greatly appreciated?

#2 Adrian K

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Posted 31 October 2010 - 05:31 PM

PstI:
CTGCA^G
G^ACGTC

ScaI:
AGT^ACT
TCA^TGA

Since I was not officially taught about doing a recombinant work, here is my thought (which is not necessary correct):
Since this is not a compatible end, you just have to cut again the 1kb fragment by using PstI, verify the fragment size, purify it and ligate it to pBR322.
Or, you can cut pBR322 with ScaI and ligate it with the 1kb fragment. (since both PstI and ScaI cut only once in pBR322 in ampicilin gene.)

Transfer to a competent cells, plate it with tetracycline plate with a sterile filter paper on top and transfer the filter paper on the next day to ampicillin plate to identify the non-growing colony which is the correct insert.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#3 Adrian K

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Posted 01 November 2010 - 10:22 PM

Ops, on second thought, I think my above post was technically wrong.

You have to treat pBR322 with ScaI, gel purifiy your fragment (remove the ~200bp fragment).
Treat your 1kb ScaI fragment with PstI, you will get 2 fragments (most probably, if your fragment have only one PstI cutting site)
Clone this 2 fragments separately into the treated vector and you will have 2 recombinant vectors.
Transform and sequence the two vector, and you will obtain the full details about the fragment of your 1kb ScaI fragment.

Edited by adrian kohsf, 01 November 2010 - 10:24 PM.

Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434




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