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Isolation of a Gene of Interest for Subcloning

Genetics as it applies to evolution, molecular biology, and medical aspects.

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Isolation of a Gene of Interest for Subcloning

Postby HawkPidgeon » Mon Apr 16, 2012 8:31 pm

Hello, everyone. I'm doing a study in genetics at my school, and I've run into a bit of a roadblock.

Part of my study involves performing subcloning. I understand that a gene of interest must be isolated from a parent vector before it can be ligated into a new vector, and that the ends of the gene of interest and the vector you're trying to insert it into have to have been cut by the same restriction enzymes.

For example, if you have plasmid A and plasmid B, and you want to take a gene from plasmid A and insert it into plasmid B, you have to do a restriction digest to cut the gene away from plasmid A, as well as a restriction digest -- using the same plasmids as you used with plasmid A -- to open plasmid B such that the gene can then be inserted.

So I'm wondering: What do you do when subcloning if your gene of interest and your vector don't have any complementary restriction sites?

Thank you.
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Re: Isolation of a Gene of Interest for Subcloning

Postby wbla3335 » Mon Apr 16, 2012 10:58 pm

Design insert-specific primers with restriction sites added to the ends that the second vector has. PCR the insert, digest the products and the second vector, and ligate.
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Re: Isolation of a Gene of Interest for Subcloning

Postby HawkPidgeon » Mon Apr 16, 2012 11:15 pm

Thank you. I've started to read about that technique, and it's exactly what I'm looking for. I was also pointed in the direction of blunt-end cloning.
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