Growing Up TA Cloning Products
landel at helios
Tue Aug 30 15:12:47 EST 1994
In article <894077843wnr at genesys.demon.co.uk>,
Duncan Clark <Duncan at genesys.demon.co.uk> wrote:
>In article: <33qe76$5rt at agate.berkeley.edu> brunet at uclink2.berkeley.edu (Lisa J Brunet) writes:
>> Andrew McArthur (AMCARTHU at UVVM.UVIC.CA) wrote:
>> : Hello everyone. For those of you that use the TA cloning kit (Invitrogen),
>> : what is your prefered method for overnight cultures of your cells with
>> : inserts? I have been using an overnight in LB (with ampicillin) but have
>> : not been getting a satisfactory amount of plasmid DNA. Has anyone tried
>> : TB media or chloramphenicol with this kit to increase plasmid DNA yield.
>> : Much thanks for your comments.
>> : Andrew
>> Yeah, me too. You'll notice that the vector is based on pBR322 so it
>> grows like crap. You can amplify it or just get enough DNA so you can
>> subclone into something else. That's what I did.
>I use TA all the time and don't have a problem with yields. It is pUC
>based with pUC copy numbers. Only if the gene is expressing and toxic
>do I have problems. Using alkaline lysis on 1.5ml O/N cultures in LB I get
>several ug plasmid. TB gives about double.
I think the problem is those sucky cells that Invitrogen sends with their
kits. Try some other bacterial strain, eg, Stratagene's XL-1 Blues
that is lacz minus.
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