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basic cloning question

basic cloning question - Protocols and Methods Forum

basic cloning question - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 07-27-2005, 03:03 AM
Sarah LaMere
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Default basic cloning question



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?

Thanks again,
Sarah


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  #2  
Old 07-28-2005, 01:40 PM
Don Incognito
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Default basic cloning question

Sarah LaMere wrote:

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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  #3  
Old 07-29-2005, 09:56 PM
Phillip San Miguel
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Default basic cloning question

Don Incognito wrote:

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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