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molecular cloning

puc19

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#1 Rampa

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Posted 11 December 2012 - 10:27 PM

I have to clone naphthalene degrading gene into e.coli using puc19 vector. i am digesting genomic DNA of my bacterial strain with Hind III and then ligating it to hind III digested puc19 (16 oC). After transformation I plate it on Nutrient agar amp plate and M9 naphthalene plate. Transformation efficiency, ligation control plates show hundreds of colonies. Nutrient agar amp plate shows many blue colonies and few white colonies, when i streak these colonies on naphthalene plate i dont get any growth, neither i get any colonies on M9 naphthalene plates after transformation.I have done this many times.
I want to know, how to achieve cloning of this gene.

#2 phage434

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Posted 12 December 2012 - 05:35 AM

So, you are actually successful in your cloning, which produces the white colonies (and perhaps a few blue ones). Your problem is that none of the clones convey naphthalene resistance. There could be many many reasons for this, few of which involve cloning. For example, the critical gene may have a HindIII site, and you may be cloning only half the gene. The gene may not express in E. coli. The gene may be toxic to E. coli. The degradation may require more than one gene. You may have sufficiently low cloning efficiency to be sampling only a small fraction of the DNA in your bacteria. You should estimate the number of required white colonies needed to sample the estimated genome size. Roughly, this would be around 5 million bases(size of genome)/4096(average HindIII fragment size)* 4(statistical coverage) or around 5000 colonies on your blue/white selection plate. You would also need to check a different enzyme to avoid the issue of cutting in the middle of a gene.

#3 Rampa

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Posted 19 December 2012 - 09:26 AM

Thanks for your response. I do have one more problem, I am getting one white colony, which is growing on ampicilin plate and naphthalene plate, but when I am staining it with gram stain, it is not like Dh5a and when I am isolating plasmid from it, it is not getting lysed in second step of alkaline lysis method.
Should I consider it as clone of my interest or should discard it as contamination

#4 phage434

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Posted 19 December 2012 - 02:20 PM

I would vote for contamination, but you might want to look at it under a microscope. I'd guess it is a yeast or other fungus.

#5 Zanetti

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Posted 21 December 2012 - 03:12 PM

1- Your blue/white screening are working fine with other transformations?
2- Is the gene in the right reading frame?
3- Have you tried a colony PCR to check the ligation?

#6 Rampa

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Posted 22 December 2012 - 07:39 PM

how can you be so sure that it is yeast or fungus?

#7 Rampa

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Posted 22 December 2012 - 07:42 PM

i looked under microscope, some cells are bigger than usual bacteria and some are small coocci/ spores.

Edited by Rampa, 22 December 2012 - 07:52 PM.


#8 phage434

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Posted 23 December 2012 - 07:07 AM

Is there a number on the size? Most yeast would be 10 u or so, most bacteria 1-2 u. But you should be figuring out what the real problem is, rather than chasing identity of some contaminant.





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