Seamless Cloning->LIC (ligation independent cloning)

Bradley Turner bsturner at MBCRR.HARVARD.EDU
Fri Aug 16 19:52:55 EST 1996


Dear Brett,

I am primarily using LIC in 3' & 5' RACE-PCR reactions, in which
I have no idea of the restriction sites that may be present in my
PCR products.  Thus, LIC allows me to clone my RACE (rapid amplification
of cdna ends) products without subjecting them restriction digestion,
and possibly cleaving internal sites. 

It also has the advantage of allowing me to clone my PCR products
directionally, which saves a little time in sequencing, expression
and probe production.

I am also using LA-PCR methods which may remove the terminal A's
of PCR products thereby reducing ligation efficiency into traditional
TA cloning vectors.

I haven't really compared the cost of adding LIC sites (11-12 nt)
to my PCR primers to that of adding restriction sites to the ends of
primers (4-8 nt *PLUS* a few extra bases to insure that the
restriction enzymes will be able to cut near the ends of the PCR
product and so that TAQ or ligase won't remove the added restriction
sites) but it seems that the cost shouldnn't be that different.

Thus for my applications, the possible added slight cost is offset
by the other potential advantages of the LIC technique.

CAVEAT:
So far I've only used LIC with positive controls and not with my
samples yet, so I would welcome any other helpful comments or 
advice regarding the LIC technique or PCR cloning in general.

Thanks,
Brad


**************************************************************
		    Bradley Turner
	         Beth Israel Hospital

Harvard Medical School		617-667-1215 phone
Division of Gastroenterology	617-667-2767 fax
Room Dana 536			turner at sprcore.bih.harvard.edu
330 Brookline Avenue		bsturner at mbcrr.harvard.edu
Boston, MA 02215		bturner at bih.harvard.edu
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To: methods at net.bio.net
From: brett at borcim.wustl.edu (brett)
Subject: Re:  Seamless Cloning->LIC (ligation independent cloning)
Date: 16 Aug 1996 14:05:40 -0700
Message-ID: <199608162104.QAA10776 at wustl.edu>

>Dear Jeanne,
>
>
>You might also want to consider LIC (ligation-independent-cloning),which
>is described in the references below, as allowing directional cloning
>of PCR products without using ligations, restriction enzymes or T/A
>vectors.
>
>Basically, one adds an extra 11-12 bases to the 5' end of your
>PCR primers. After PCR, the PCR product ends are digested in the 
>presence of T4DNApol with dTTP or dATP which exposes the 11-12
>LIC site at the ends of your PCR product. This is *annealed*
>with the LIC vector at RT for about 30min and then transformed
>into e coli (NO ligations, NO restriction digests).

Maybe you could comment on the cost effectiveness of this method. As you
describe it, you'd be adding an aditional $20 in nucleotides per PCR just to
clone the thing. I have never had too much difficulty in standard RE cloning of
PCRs, so I'm not convinced of the utility of LIC.



Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             

"I own my own pet virus. I get to pet and name her." - Cobain




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