adaptors for cloning - how it works? (Sep/07/2006 )

HI I heard we can us adaptors oligomere for cloning. How it works?

any example?

thanks

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do u mean like a linker where u would order 2 oligos with the corresponding sense and antisense and annealing them and ligate into a vector ?

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http://www.protocol-online.org/forums/inde...showtopic=19650

I made a nice drawing of an adaptor. Please visit this post.

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hi
I mean for exemple:
I have a ecoR1 that i want to be able to clone in Bamh1.
how will look the adpater?
how long it should be etc...


thanks

jm

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This ( AATT GGATCC ) adapter will convert an EcoRI site to a BamHI site. This adapter will also kill the EcoRI site. This adapter is 10bp long. As this adapter is palidromic, only a single oligo need be purchased.

An adapter can be made to introduce more then 1 Restriction site, allowing orientation of the insert which willl use the adapted site.

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/thanks you very much.
so I just need then to cut with EcorI and ligate my adaptator right?
But I may end up with concatemer adaptator after ligation right?

thanks

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yes, concatemers are possible, (though rare) if the linker has 5' phosphorylated ends (added by either the nucleotide company at extract cost or by treatment with PNK). 5' phosphorylation makes ligation easier though..

However, since this is a single restriction site linker, concatemerisation is not a problem. The restriction enzyme will still cleave the linker (on nearly every copy) which will still leave BamHI ends.

For a small linker like this, (and thus high mol production and hence high molar concentration) i don't bother with 5'end phosphorylation, thus by passing the concatemer problem.

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