Ligation problems

[Ned Mantei]

In article 
<A5F19D5320D79440B64D622955C15F33096784 at somaserver1.sladmin.com>,
 tfitzwater at somalogic.com (Tim Fitzwater) wrote:

>"Why do I have so many white colonies that result in a plasmid with no
>insert?" (Stratagene FAQ at their website)
>"It is imperative that you use at least a 40:1 insert:vector ratio in the
>ligation. Too low an insert concentration will result in false positives due
>to the increased enzymatic activity and constant cutting and religating of
>the vector ends. Bases can be lost in this situation causing a frameshift
>which results in a white colony."
>
>Tim Fitzwater
>SomaLogic
>---

If the reaction in question is "normal" ligation, with ends generated by 
restriction enzymes and joined by DNA ligase, you might want to look at 
the article cited below. For vector treated with phosphatase, the best 
results will typically be with ca. 5 or 10 fmol of vector and 10 fmol of 
insert in ca. 10 ul--a molar ratio of insert to vector of ca. 1:1 or 2:1.

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Nucleic Acids Res.  1988 Nov 11;16(21):10301-21.  

Kinetic analysis for optimization of DNA ligation reactions.

Revie D, Smith DW, Yee TW.

Department of Biology, University of California, San Diego, La Jolla 
92093.

Kinetic equations describing ligation of DNA to circular recombinant 
forms were
developed and solved for four types of reactions: (a) a homogeneous 
population
of singly restricted DNA fragments, (b) insertion of singly restricted 
insert
into vector, (c) forced directional insertion of doubly restricted 
insert into
vector, and (d) insertion of singly restricted insert into phosphatased 
vector.
The effects of varying vector and insert sizes, starting concentrations, 
and
phosphatase treatment on the yield of circular 1:1 recombinants were 
analyzed.
Selected theoretical predictions were experimentally tested and 
verified. Our
suggestions on optimizing ligation reactions in several cases are at 
variance
with common practice. For example, optimum conditions in case (b) and (d)
ligations are best specified as individual insert and vector 
concentrations
rather than as insert/vector molar ratios, except in case (d) ligations
involving very small insert size. In case (c) ligations, highest 
efficiencies
are obtained when both vector and insert are at relatively low 
concentration.

PMID: 2848221 [PubMed - indexed for MEDLINE]
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-- 
Ned Mantei
Department of Cell Biology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland