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How to determine if DNA fragment is in a plasmid?

How to determine if DNA fragment is in a plasmid? - DNA Techniques

How to determine if DNA fragment is in a plasmid? - Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


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Old 08-03-2009, 07:16 PM
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Default How to determine if DNA fragment is in a plasmid?



I have inserted a DNA fragment into a plasmid. The plasmid has then been transformed into E.coli which has then been grown. I have confirmed that the bacteria contains the plasmid, but I want to be sure that the plasmid contains the DNA fragment, how do I do this?
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Old 08-03-2009, 07:16 PM
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The plasmid might've had some important function.

So you should test for the function. By inserting a DNA fragment into the plasmid you might have inactivated the function of the plasmid.

If you have a plasmid that included like a lacZ gene you could insert your fragment of interest directly into the lacZ gene and inactivate the lacZ gene

Provided that you used the correct restriction enzymes and restriction sites.
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Old 08-04-2009, 01:22 AM
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Default Re: How to determine if DNA fragment is in a plasmid?

Alternatively, you could do sequencing.
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Old 08-04-2009, 01:37 PM
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Default Re: How to determine if DNA fragment is in a plasmid?

1) sequencing
2) pcr of your plasmid (with primers to your insert or with sequencing primers of your plasmid). if you have pcr-product with expected size, you have insert.
3) restriction. in gel after phoresis the restricted plasmid with insert looks different from restricted in the same way plasmid without insert
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