basic cloning question

Don Incognito imperat at hotmail.com
Thu Jul 28 08:40:59 EST 2005


Sarah LaMere wrote:
> OK, so you guys helped me amplify my 4 kb fragment from cDNA.  Now I just need to clone it, and it's not cooperating.
>
> I'm using Elongase to amplify my product, which includes a proofreading enzyme.  I've been adding A overhangs to my gel product (purified with a millipore kit) and then cloning using the Invitrogen topo TA cloning kit.  It's basically just not working.  Any suggestions for cloning large inserts?

Right off the top, your major mistake is trying to topo-clone a 4kb
fragment. In my experience, Topo is best used with tiny things. Your
best bet would be to use the old-fashioned (restriction) digest,
(alkaline phosphatase) dephosphorylate and (T4 DNA ligase) ligate
method. I assume you have restriction sites placed for directional
cloning this way?

Don Incognito
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