Help - cloning into dephosphorylated plasmid vector

Hiranya Roychowdhury hroychow at NMSU.EDU
Thu May 25 15:26:14 EST 1995


On 25 May 1995, Aron B. Jaffe wrote:

> Nick dos Remedios <N.dosRemedios at uts.edu.au> wrote:
> >
> > In a nutshell: I am unable to ligate foreign DNA into the ECORI site of
> > my plasid vector (pcDNAI) after the vector cut with EcoRI then treated
> > with C.I. alkaline phosphatase. Cut vector re-ligates back together quite
> > efficiently, therefore the problem seems to be the CIAP step but I have
> > been unable to fix it [I *think* my overhangs are being eaten by the
> > CIAP].
> > 
> > The result I get after ligating (O/N at 16 degC) a test insert (EcoRI
> > ends) of 1.7 kb and transforming into my bugs (MC1061/p3) is that I get
> > background numbers of colonies for all ligation reactions (background =
> > no insert control ligation reaction). i.e. trying 3 different molar
> > ratios of vector to insert (3:1, 1:1, 1:3).
> > 
> > CIAP treatment: I am using Promega CIAP molecular grade and have been
> > following their protocol. I have tried 2 different batches of this
> > enzyme. Cleaning up after the CIAP reaction seems to be critical and I
> > have tried the Promega method and both of the Sambrook et al. methods
> > (heat denaturing enzyme & digesting with proteinase K; followed by
> > phenol/chloroform extraction etc.). I have also tried heat denaturing
> > followed by GeneCleanII DNA clean-up and purification. NONE of these
> > measures have made any difference!
> > 
> > The only suggestion I have received from someone who has used this
> > vector, is, forget the CIAP step (i.e. don't dephosphorylate vector) or
> > else clone into two separate R.E. sites (directionally clone). However, I
> > already have cDNA adapted with EcoRI so I don't want to waste it.
> > 
> > If you have any suggestions please reply/post, I am desperate!
> > 
> > Nick
> > 
> > Nick dos Remedios                                Immunobiology Unit, UTS.
> > N.dosRemedios at uts.edu.au                         tel (02) 330 4045
> 
> I agree with your friend - forget dephosphorylating the ends...
> there's no need to do that with sticky end ligations...
> 
> aron
> 
> 

That is not quite correct, what you said there, Aron. In fact, 
dephosphorylation should only be left out if the vector is cut with two 
enzymes generating non-compatible ends. When the vector is digested with 
EcoRI only, self ligation is favored, unless it is 'CIP'ed. 
	Back to the original problem. CIP is a difficult enzyme to get 
rid of and may ruin the ligation. So, here is the way I prepare my vector 
following CIPing:
	I heat the CIPed vector at 70 C in the presence of 10mM EDTA and
1% SDS, for 15 min (some people even heat it to 80 C for 10 min). Then I
let the DNA cool to RT and extract it with PCI three times, followed by
two CI extractions. I electrophorese this DNA on a low percentage agarose 
gel (preferably LMP), and collect the DNA off that using DEAE membrane 
(some people call it "tombstone" method). Glass milk is also fine, as 
along as you are careful not to shear the vector. Using the above method, 
I have never had any trouble with my ligations involving CIPed vectors. 
In fact, I never use phosphorylated vectors.

			>>>>>>>>>>>>>>>>>>>>>>>>>>>
			  Hiranya S. Roychowdhury
   			  Plant Genetic Engineering Lab.
			  Box 3GL, NM State Univ.
			  Las Cruces, NM 88003
			  Phone: (505) 646-5785
			  hroychow at nmsu.edu
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