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plasmid isolation problem

plasmid isolation problem - Molecular Cloning Forum

plasmid isolation problem - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


View Poll Results: plasmid isolation problem
Is the colonies of dh5 alpha appaered as pink? 0 0%
is there any different protocol for plasmid isolation from dh5 alpha. 0 0%
transformation efficiency of xl1 blue cells. 0 0%
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  #1  
Old 03-25-2011, 07:53 AM
Pipette Filler
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Question plasmid isolation problem



Hi,

I am facing one problem with plasmid isolation. nw i am doing one project wrk on pcr mutagnesis.

transformation of the pcr product gave one colony after 12 hrs. i isolated the dna from the colony( XL-1 blue supercompetent cells were used for transformation)
But the concentration is very less, i.e; appro.. 25ng/ul

so i again transform the isolated plasmid to DH5 alpha cells to get more.

But it also gives lowest concention.I have done these things repeatedly.

I am using the phenol chloroform method for the plasmid isolation.According to the competent cells used,is there any change in the protocol..

please help me, if i didn't get enough plasmid, i am not able to send it for sequencing.


please.......................
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Old 07-29-2013, 10:24 AM
Pipette Filler
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Default Re: plasmid isolation problem

i have read your query about pcr mutagenesis.
Even i am facing the same problem in Site directed mutagenesis.
After getting colonies, I am not able to isolate plasmid from XL1-Blue transformed supercompetent cells and even not able to transform it further in DH5a E.coli cells.
Please suggest me as i have stucked in between and not able to give samples for sequencing.
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