Tripple Ligation - (Jan/11/2005 )

Hi all

I'm trying to ligate three components:
1. A 3KB vector cut with BamHI-Xhoi
2. A 0.9 KB fragment cut with BamHI-SacII
3. A 0.8KB fragment cut with XhoI-SacII

I tryied diffrent mollar ratios and did not get any positive colonies.
What am I doing wrong???

Thanks,
Anat

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Hi,

i found that my ligations are working better, when I heat the ligation sample to 65°C for 5 min. I let the solution slowly cool down and than add the ligase to it. Following incubation overnight at RT

I never tried a tripple ligation, but it might be helping you as well

good luck
MineX

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This paper may be helpful for your ligation.
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Molecular cloning is a crucial, and often speed-limiting, step in
many standard procedures of molecular biology. Ligation of
cohesive DNA ends is normally carried out at 12¨C16 C to ensure
a good balance between enzyme activity and stability of annealed
DNA overhangs. Low temperatures generally reduce ligase
activity, whereas too high temperatures may reduce cloning
efficiencies by melting annealed DNA overhangs and increase
overall molecular motion in the ligation reaction. Several
procedures have been described to increase the efficiency ligation
reactions, including the addition of condensing agents as
polyethylene glycol (1) or hexamminedicobalt chloride (2).
These approaches induce macromolecular crowding, and thus
serve to mimic higher DNA concentrations in the ligation
reaction. Other approaches seek to increase the efficiency of
molecular cloning procedures by omitting the ligation step by
generating long single-stranded DNA overhangs that can be
annealed and transformed directly into an appropriate Escherichia
coli host (3). Ligation of blunt-ended DNA fragments is normally
carried out at room temperature using higher concentrations of T4
DNA ligase.

We have devised a simple procedure in which high enzyme
activity and DNA annealing is balanced by constant temperature
cycling. We find temperature-cycle ligations (TCL) may increase
the efficiency of staggered cut cloning ~4¨C8-fold, while the
efficiency of blunt-end clonings are increased ~4¨C6-fold.

The bacterial cloning vector pBluescript II KS+ was digested
with AflIII and HindIII producing two cohesive end-fragments of
434 and 2526 bp respectively, or PvuII generating two blunt-end
fragments of 448 and 2512 bp respectively. All enzymes were
purchased from Amersham. Digested plasmid DNA was separated
on 1% low-melting agarose (NuSieve) and purified as
described by Sambrook et al. (1). For each digestion a ligation
master mix was prepared containing 50 mM Tris¨CHCl (pH 7.6),
5 mM MgCl2, 5 mM DTT, 50 mg BSA/ml, 0.5 mM ATP, 200 ng
DNA and 0.1 Weiss unit T4 DNA ligase/10 ml. The purified
fragments were added in equimolar amounts. The master mix was
divided into 10 ml ligation reactions and either incubated at 14 C,
at room temperature (22C), or subjected to temperature cycling.
Temperature-cycle ligations were carried out for 12¨C16 h in a
Perkin-Elmer-Cetus DNA Thermal Cycler 480 programmed
indefinitely to cycle between 30 s at 10 C and 30 s at 30 C. In
our setting, the machine performed 10 cycles/h. Competent
XL1-Blue E.coli cells were transformed by electrotransformation
according to manufacturer¡¯s directions (BioRad), transferred to
1 ml LB medium and incubated at 37C for 1 h. For enumeration
of cloning efficiencies aliquots of 25¨C100 ml were spread onto LB
plates containing 50 mg/ml ampicillin. The number of colonies
appearing per transformation is shown in Table 1. We have tested
the kinetics of temperature-cycle ligations over 12¨C16 h, and
found them to be similar to those of ligations at constant
temperatures resulting in a linear increase with time in the number
of colonies appearing after transformation (data not shown).
Furthermore, temperature-cycle ligation may be used together
with hexamminedicobalt chloride in blunt-end ligations to give
the combined effect.

Temperature-cycle ligation is routinely used in our laboratory,
and, we believe the technique to be broadly applicable in all
protocols involving molecular cloning, with special relevance to
difficult clonings and library construction.

(Nucleic Acids Research, 1996, Vol. 24, No. 4)

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Hello
Can you tell us if the triple ligation worked? We are trying the same thing too. Our pieces are 8kb 40b, and 760 b.
Any help is greatly appreciated
AJ

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I am doing the similar experiment as well....

Did your triple ligation work??

This is mine problem

http://www.protocol-online.org/forums/inde...?showtopic=7281

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I met the same problems as yours.

Did you try to ligase your two fragments (2. A 0.9 KB fragment cut with BamHI-SacII 3. A 0.8KB fragment cut with XhoI-SacII ) together first and then try to extract the linear ligation , then try to insert it to the vector ??

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Hi,
I am also facing problem with my triple ligation of

vector fragment :6.5kb
insert 1: 1.3kb
insert 2:.0.3 kb

I tried triple ligation as well as two step ligation (where I am ligating the two insert fragments first).

I am not getting any true colonies.

Can anyone help me with suggestions.

Thanks in advance
[indent][quote]

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One option we have used (though only useful if you have overlap between the insert pieces or are using PCR primers to design the pieces yourself) is to do a variation of molecular breeding, fusing the insert pieces by PCR. Design the 2 or more pieces to have sufficient overlap to anneal and simply put the 3 products together in a PCR reaction with these modifications 1) use no primers, the three pieces prime each other, 2) use a long (5') initial denaturation, 3) run only about 5 cycles. (4. use high fidelity polymerase for expression cloning). This should allow the three to inter-prime and fill in the complete fusion product. We have done it with up to three pieces at once to make a 1.4kb insert. I am attempting a 3 piece 4.5kb insert this way for expression cloning and can update in a month or so.

Then run a second normal PCR on the above using the first piece's forward primer and the final piece's reverse to amplify only the complete target fusion product. Only the final forward and reverse primers need restriction sites added. Then you can ligate into a vector as a single insert - simplifying the cloning. Theoretically you could even PCR the vector into the product by designing the outer primers properly so long as complete product is within the PCR length limits and then recircularize before transforming - but I have never tried that.

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