Summary: Plasmid Yield from TA Cloning Kit
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.
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.
More information about the Mol-evol