Question on Plasmid Cloning

John R. McQuiston zje8 at cdc.gov
Tue Aug 10 07:14:01 EST 1999


I'm assuming that you mean a Bam---Hind^Hind--Bam^Bam--Hind if you are ligating 
into a double cut vector.  This does occur but very rarely.  I have seen it 
though.  Usually it is selected against for 2 reasons, size of the insert and 
complexity of the multiple ligation.  It is easily resolved if you do get one 
though.  Just digest with Hind (or Bam) alone take 1ul of the digest and go 
straight to ligation.  The dilution effect will select religation of the vector 
containing only one insert.  


John




In article <7oouq1$iha$1 at oyez.ccc.nottingham.ac.uk>, mucineer at iname.com says...
>
>Dear All
>
>As a new comer to molecular biology, I have the following trivial question
>and hope someone can help:
>
>When ligating a DNA insert into a plasmid (both the insert and plasmid
>contain different restriction sites on either ends, e.g. a BamHI----HindIII
>fragment into a vector digested with these two restriction enzymes), is
>there anything in the process that prevent different molecule of the insert
>from "self-ligating" to form species such as
>HindIII-------BamHI--------HindIII or BamHI--------HindIII------BamHI?
>
>This should be a common problem to all ligations but it doesn't seem to be a
>big problem as I usually get hundreds of colonies on the plate.
>Does anyone have any insights into this?
>
>Any comments will be appreciated.
>
>
>
>
>




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