Why is Ligation of Oligo A and Complement B Only Producing Monomers and Dimers?

[karthik3k]

Somebody please explain ...

I have an oligo (A) (non-phosphorylated in 5') of ~ 100 bases.
I have its complement (B) with two over hangs (~4 bases each) on both he sides(5' and 3').

the overhangs on the B are palindromic. as in the 5' overhang is palindromic to itself.

I annealed both of them.
phosphorylated the sample.
added ligase


after ligation, when i ran it on the gel, i saw only monomers and dimers.

Why is this so ?
y not other multimers ?

did i go wrong sumwhere ?

please help

 

[iansmith]

I think the problem is that your B oligo has both overhang. I would have an overhang on each oligo so they annealed as follow (the overhang are in bold)

[B]GCTA[/B]GCTGCAAAGCTGCTGC
    CGACGTTTCGACGACG[B]CGAT[/B]

Also what is the annealing condition? You temperature might the optimal for annealing your primer.
What kind of ligation process are you using? 4 C overnight or 25C for 1 hour. The overnight incubation increase the yield and the number of different products.
Did you heat your DNA at 65c prior to adding your ligase and ligase buffer? This increase the odds of DNA coming together properly.

 

[karthik3k]

the sequences are like :

Seq A XX---XX

Seq B GATCXX---XXCGAT

so theoritically,
they sud form like ...
------XX---XXGTACXX---XXCGATXX---XX
GATCXX---XXCATGXX---XXGATCXX---XXCATG

and so on...

But it didnt !
ligation was carried out at 25 deg for over night.
sample was kept at 65 deg before adding ligase!

 

[iansmith]

the sequences are like :

Seq A XX---XX

Seq B GATCXX---XXCGAT

so theoritically,
they sud form like ...
------XX---XXGTACXX---XXGATCXX---XX
GATCXX---XXCATGXX---XXGATCXX---XXCATG

and so on...

But it didnt !

Hopefully you made a mistake will typing the text because your overhangs are not complementary.

In theory, it should work but it is not always the case and having both overhang on the same oligo might make the physical interaction more difficults.

ligation was carried out at 25 deg for over night.
sample was kept at 65 deg before adding ligase!

That may be a stupid question but did you cool the sample prior to adding the ligase. Ligase is inactivated at 65C.
The rest of the ligation appears to be ok.

What are you annealing temperature and condition?

 

[Monique]

first of all, the ends are not complimentary.

karthik, which ligase are you using and how fresh is it? especially when the enzyme is atp dependent, you should make sure it's fresh since atp is prone to degradation.

why do you use the phosphorylation step, can't you omit it?

 

[Monique]

I also see a problem in the annealing step of the different oligos. It might be time consuming, but won't it be more efficient to clone the fragment of interest into a vector, grow it in e.coli and cut it out with restriction enzymes?

That way you are absolutely sure that you have a double stranded piece of DNA with sticky ends.

 

[karthik3k]

Sorry, I corrected it

------XX---XXGTACXX---XXCGATXX---XX
GATCXX---XXCATGXX---XXGATCXX---XXCATG

Annealed it at 37 deg for 3 hrs.

I can clone it, but what i need is a tetramer...
But i get only monomer and dimer :(

 

[Monique]

so how sure are you that your enzyme is fresh and why are you doing the phosphorylation step

 

[iansmith]

Annealed it at 37 deg for 3 hrs.

That might be the problem. Your annealing temperature is too low.

I usully mix equimolar ratio of the primer in TEN Buffer (10 mM Tris, 1 mM EDTA, 0.1 mM NaCl, pH 8.0 with HCl). I then incubate the primer from 95C to 4C over 1 hour. I usually do this in a thermocycler.

This might also be a suitable protocol

I mix the oligos in an equimolar ratio say 50 ul of 100uM solution of each. Bring the volume to 294 ul with water and heat in a block to 85°C for 10 min. Add 6ul of 5M NaCl and back into 85°C block for 10 min. Turn off the block and allow to cool to room temp. Once at RT ppt. with 30ul 3M NaOAc, 750ul EtOH. Chill on ice and spin at 4° for 15 min. Rinse with 70-90% EtOH and resuspend in TE.
To determine whether they annealed for oligos as long as yours simply run a sample of each of the unannealed next to the annealed oligos on a 3% agarose gel. You won't get sharp bands, more like blobs, but the annealled will run as a larger band (double vs. single stranded). I just did this for a 48 and 55 bp pair of oligos, worked like a charm.
To determine concentration I would just use a spec. and for ligations I use a large excess of annealed oligo to vector, 10:1 or more seems to work well.
Good Luck!

http://micro.nwfsc.noaa.gov/forums/viewtopic.php?t=10003&highlight=oligo+annealing

 

[karthik3k]

the dna that i use is of pnly ~ 100 bases. n its melting temp is 71 deg.

Yes.
I used fresh enzymes (NEB). i phosphorylated, since ligation needs phosphorylated ends ...
The oligos provided do not have phosphate grp at 5'

I first annealed the oligos at 37 deg for 3hrs.
added T4 ligase buffer (since it has ATP).
added PNKinase. left it for 1-3 hrs.
deactivated at 65 deg for 10 min.
then added T4 ligase and left it overnight at room temperature...
the reaction volume is 15-40 uL.

sud i increase the annealing temp ?
sud i decrease ligation temp ??
is the reaction volume very low ?

watever the mistake may be...
y cudnt i see even a faint band of multimers ?

 

[iansmith]

You have to increase your annealing temperature. I should be around 70C. 37C might created unspecific binding and it is suboptimal for binding. I suggest that you follow either protocol I gave you above.

The rest is ok but you should not heat you ligase buffer to 65C. You should add the buffer after inactivation of the enzyme.

 

[Monique]

karthik, did you try and increase the concentration of your oligos that you use during ligation? what concentration are you currently using?

 

[Monique]

http://www.plantphysiol.org/cgi/content/full/134/3/890

might have a usefull protocol, concatamers are used in LongSAGE

 

[karthik3k]

i have varied concentrations of the DNA. but of no use ...

Can u temme wat happens to the buffer when it is heated to 65 deg ??
will the ATP degrade ??/

and i think i can't use 70 deg for annealing temp, coz the melting temp is 71 deg.

 

[Monique]

i have varied concentrations of the DNA. but of no use ...

between which concentrations did you vary it? you really need to know with what concentrations you are working.

 

[Monique]

Ok, I looked up my experiment. This is what worked for me:

9.0 ul DNA 210 bp [184 ng/ul]
1.05 ul 10x ligase buffer
0.5 ul ligase 3 U/ul

I did a number of experiments to try and optimize the longer concatamer formation. What I found is that at timepoint 15 minutes, the reaction has already come to an end. Not sure why, most likely circular product formation. On gel I typically get a ladder of 6 bands and a smear on top where the fragments are too large to be separated.

The reaction also worked for DNA of 100 bp. I prepared the DNA by cutting it with restriction enzymes out of a vector, after which it was agarose gel purified.

 

[karthik3k]

500ng/uL - 1ul, 2uL ... of oligo
1000ng/uL(1 ug/uL) - 1ul, 2uL ... of oligo

BTW,
u still didnt temme wat wud happen if one heats the buffer to 65 deg ...

 

[iansmith]

Can u temme wat happens to the buffer when it is heated to 65 deg ??
will the ATP degrade ??/

I don't know what happen per se but from personal experience, putting the ligase buffer at 65C always decrease my efficiency.

and i think i can't use 70 deg for annealing temp, coz the melting temp is 71 deg.

The optimal annealing temperature is probably around 68C. I have also used annealing temperature above the melting temperate and it gave good results.

The idea would be heat you oligos to 95C so they denature then cool let it cool down to room temperature so the the oligo anneal at the optimal temperature without you pick a temperature. That the principle in the two protocol i gave you.