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ligation - very small insert - problems during ligation (May/28/2008 )

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I digested plasmid pHannibal with 2 different restr. enz., that gives me 3kb fragment with 1 blunt and 1 stick end. My insert is very small (about 67bp) and is made by 2 ss oligonucleotides hybridization (syntetized as primers; after hybrydization I obtain ds oligonucleotide with appropriate ends - the same ends will occur after digestion). The problem is during ligation this small insert into digested fragment of my plasmid. Did anyone do similar ligation? I tried several times with diffirent molecular ratio, and still without results. Is it possible to ligate so small fragment? What do you suggest?
thanks for any helpful answers

-popdok-

One key element you may be missing is phosphates on the 5` end of the oligos. It was a good idea to create the sticky ends in your design but standard oligonucleotides do not come phosphorylated so unless you've ordered them with phosphates or added them yourself the ligation will not work (assuming you've dephosphorylated your vector).

-killerkoz17-

I agree that your oligos need to be phosphorylated. When doing a sensitive ligation like this, I'd also make sure that my vector was 100% linear (gel purification is a must) and very thoroughly dephosphorylated.

When I was preparing vectors for making cDNA libraries (which is also very sensitive) I'd even test my vector in a set of reactions with:

1) a longer insert with appropriate sticky ends (for example, just find a plasmid that generates a 1 kb fragment when cut with your enzymes, gel purify the fragment and use it as an 'insert')
2) an insertless self-ligation reaction (negative control)


After plating 20 and 200 ┬Ál of the ligation reaction on plates, if the vector is good, the positive control plates should have lots of colonies and the negative control plates should be empty or nearly empty. I would set up a similar set of test ligations when ligating in a linker. If you plan it correctly in advance it should only take an extra day (because you can digest and gel purify both the linear vector and positive control insert DNA in parallel).

Best of luck,

Ginger

-Ginger Spice-

The smallest the insert is the easier the cloning should be wink.gif One little risk is the concatemer formation (cloning of multiple copies of your ds iinsert) that's the tricky part

-Jipes-

thanks for good advises wink.gif it's totally true about not phosphorylated oligos. Maybe it is strange but I did very similar ligation 3 years ago (using the same method; without phosphorylated ds oligos) and everything worked fine that time. Will biology ever stop suprising me ? wink.gif

-popdok-

Biology never stops surprising.

As for my two bits;
I don't use phosphorylated oligoes (it cost more money).
I find ligation is easy if you flood the ligation mix with the linker. So to the digested vector, added as high concentration of linker as you can.

typically for me it means something like
2ul vector
15.5ul 10uM linker (in water)
2ul ligase buffer
0.5ul T4 ligase.

And what every ligation method you do chose to use, a kill digest is useful. If upon ligation of the vector to the oligo, the restriction site is destroyed, a kill digest can be used to remove religated vector molecules.

-perneseblue-

QUOTE (perneseblue @ May 30 2008, 11:40 AM)
Biology never stops surprising.

As for my two bits;
I don't use phosphorylated oligoes (it cost more money).
I find ligation is easy if you flood the ligation mix with the linker. So to the digested vector, added as high concentration of linker as you can.

typically for me it means something like
2ul vector
15.5ul 10uM linker (in water)
2ul ligase buffer
0.5ul T4 ligase.

And what every ligation method you do chose to use, a kill digest is useful. If upon ligation of the vector to the oligo, the restriction site is destroyed, a kill digest can be used to remove religated vector molecules.

Jeepers, that's a like a gazillion molar excess. And you're relying on phosphorylated vectors floating around there aren't you? Where are the phosphates?

popdok, you can phosphorylate them yourself using T4 PNK instead of buying them phosphorylated, although for one offs why not?

As for concatemers, dilute your ds insert around 1:100 to 1:1000 before ligation and use 1 uL in the ligation. That will help avoid the high molar excess favouring concatemer formation.

-killerkoz17-

QUOTE (killerkoz17 @ Jun 2 2008, 12:30 PM)
QUOTE (perneseblue @ May 30 2008, 11:40 AM)
Biology never stops surprising.

As for my two bits;
I don't use phosphorylated oligoes (it cost more money).
I find ligation is easy if you flood the ligation mix with the linker. So to the digested vector, added as high concentration of linker as you can.

typically for me it means something like
2ul vector
15.5ul 10uM linker (in water)
2ul ligase buffer
0.5ul T4 ligase.

And what every ligation method you do chose to use, a kill digest is useful. If upon ligation of the vector to the oligo, the restriction site is destroyed, a kill digest can be used to remove religated vector molecules.

Jeepers, that's a like a gazillion molar excess. And you're relying on phosphorylated vectors floating around there aren't you? Where are the phosphates?


T4 ligase buffers usually come with ATP in them.

-swanny-

QUOTE (killerkoz17 @ Jun 2 2008, 02:30 AM)
popdok, you can phosphorylate them yourself using T4 PNK instead of buying them phosphorylated, although for one offs why not?


thanks for advice wink.gif but I would rather dephosphorylate my digested vector than buy expensive phosphorylated oligos wink.gif

-popdok-

QUOTE (popdok @ Jun 2 2008, 07:17 PM)
thanks for advice wink.gif but I would rather dephosphorylate my digested vector than buy expensive phosphorylated oligos wink.gif

That didn't make sense, but i understand you dont want to buy the phosphorylated oligos (really not that expensive). Any way, dephosphorylating the vector must be done to prevent background colonies. I would phosphorylate your oligos or ds insert using T4 PNK to get the phosphates for ligation.

I think i see how what Pernese is talking about will work. No phosphates present anywhere and relying on the attraction between the sticky end bases to hold the insert in the vector and then letting the bacteria to repair the nicks? Is that right Pernese?

Swanny, I was referring to phosphates on the DNA required for ligation, so i dont think the ATP in the buffer has anything to do with what i was saying.

-killerkoz17-

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