basic cloning question

Phillip San Miguel pmiguel at purdue.edu
Fri Jul 29 16:56:01 EST 2005


Don Incognito wrote:
> 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
> Insert "spam" to reply by e-mail.
> 

We use pCR4TOPO cloning kits to make 4-8kb average insert-size shotgun 
libraries from BAC DNA. Works fine. In fact, seems like one only begins 
to run into difficulties above 12 kb inserts.

Your PCR primers are not 5' phosphorylated, are they? If so, that would 
inhibit the topo reaction. But it is unlikely you are using 5' 
phosphorylated primers.

Maybe skip the A-tailing and use pCR4TOPOblunt?

Another possibility--during gel purification are you frying your band 
with UV? Better to use a Dark Reader for stuff you are going to clone. 
If you don't have access to one, minimize exposure of your DNA to the UV 
and use a long wavelength UV box, if possible.

Also, avoid borate in your agarose gel buffer. TAE can be used instead 
of TBE. The millipore should get rid of the borate--but who knows. 
Borate can lower transformation efficiency.

Finally, what do you mean by "basically not working"? You get no 
colonies? The plasmids don't have inserts? Something else?

-- 
Phillip SanMiguel
Purdue Genomics Core Facility


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