Summary: Plasmid Yield from TA Cloning Kit

Andrew McArthur AMCARTHU at UVVM.UVIC.CA
Thu Sep 1 14:34:25 EST 1994


Hello.  Recently I posted a query about culture of cells from the
Invitrogen TA cloning kit such that I could yield large amounts of
plasmid DNA.  Previously I had been disappointed when using simple
overnights in LB media.  Here are the various suggestions I
received.  Much thanks to all of those who replied.

Andrew


1. Switch to Promega's pGEM-T kit (cheaper, grows great).  Quite a
   number of people suggested this.

2. Subclone it into something else (as the kit's vector is based on
   pBR322 which grows poorly).

3. Make your own vector by cutting Bluescript (or whatever you
   want) with a blunt end cutter and tailing with Taq and dTTP (see
   Nucleic Acids Research 19: 1154)

4. Use kanamycin (despite the cost) instead of ampicillin with the
   TA cloning kit as it reduces satellite colonies and minipreps
   are loaded with plasmid DNA. [Note: the TA cloning kit vector
   has both ampicillin and kanamycin resistance genes]

5. Terrific Broth (TB, see Maniatus et al.) with ampicillin works
   much better than LB media for the TA cloning kit cells.

6. Do not use the Invitrogen cells (some researchers are not happy
   with them) but try some other bacterial strain.



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