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ligation - stick 3 pieces together


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#1 postdoc

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Posted 15 September 2004 - 08:22 AM

Hello,

I want to clone a artifical fragment of about 100 bp into a vector. Maybe it is too long to synthesize 100 bp oligo, so I break the sequence into halves and designed two pairs of oligos for each of the halves (about 50 bp). The two pairs have stick end so they can be stuck together to make the 100 bp fragment. They also have stick ends corresponding to the vector restriction sites.

My question is:
if I put the two pairs of annealed oligos and vector in one ligation reaction, what is the chance I success? Or should I ligate the two ds-oligos first and then insert them to the vector?

Thank you very much for your help.

#2 biotech

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Posted 17 September 2004 - 10:59 AM

Why not try and see? It seems to me that either way would work, but I've never done that kind of stuff. By the way, it is possible to synthesize polynucleotides over 100 bp, so why wont you use this way instead?

#3 postdoc

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Posted 17 September 2004 - 05:48 PM

Thank you, biotech,

The reason why I didn't synthesize 100 nt oligos is that my sequence contains 4 copies of a repeat element and I was afraid that the sense and the antisense strand would anneal half way from each other instead of from end to end.

I am in the process of doing this experiment and will keep you posted. I have annealed the oligos to form 2 pieces of ds-oligos and checked by running a gel. ds-oligos looked differently compared to single-stranded. then I cut my plasmid with BglII and KpnI, and did a purification. Then I did a ligation with the two pieces of ds-oligos and linerized plasmid in a 10 ul volume. After that I ran a gel using 4 ul of the ligation reaction. Strangely, ligation without insert also show circular vector which is a little bit smaller than ligation with insert. So I figured that my two pieces of ds-oligos have been inserted to the vector. However I have no idea why my linerized vector got re-circularized too because the two ends are not compatible. Anyway, I did a transformation, and have just got miniprep done and am doing restriction to verify if my insert is there...

#4 postdoc

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Posted 18 September 2004 - 09:43 AM

Updates on my experiment

I have minipreped my plasmid and cut with Bgl II/KpnI and ran a gel. Some plasmids have been linerized but some remained intact. I didn't see any plasmid release 100 bp insert after cutting.

One potential problem is the enzymes I used because Bgl II/KpnI are not compatible in double digests. I will repeat it by doing a sequential digests.

My question is Should I be able to see the 100 bp insert on the gel if it has been cut from the vector?

#5 kant0008

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Posted 19 September 2004 - 08:34 PM

Hey postdoc,
100bp is large enough to see, all depends on the concentration of your vector on the gel. But if you get a nice bright band for the vector, then yes, you'd expect at least a very faint band for 100bp fragment

#6 postdoc

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Posted 22 September 2004 - 03:09 PM

Update on my experiment:

I cut my vector and could not see the insert, so i have to start over.

Althogh NEB says that KpnI and BglII can be used in double digests but I tried without success. My plasmid was not linerized even after o/n cut with newly ordered enzymes. So I have to do sequential cuts first with KpnI and then BglII.

I am going to do the ligation again. My question is whether it is possible to check if my 100 bp insert has been ligated into my vector before transformation. I can run a gel using ligation reaction along with the original vector (non cut, no insert), but the difference is only 100 bp, I doubt I can see the difference. Any suggestion is appreciated.

#7 kant0008

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Posted 22 September 2004 - 04:13 PM

hi again,
as far as I understand your cloning is directional, you are using 2 enzymes ? In that case if you do a ligation control with cut plasmid only it should not religate. So then if you run that alongside your annealed product it should run differently if your 100bp fragment is ligated, as ligated plasmid should run higher (normally) thatn cut.
Hope that helps

#8 postdoc

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Posted 22 September 2004 - 04:19 PM

Thank you very much, Kant.

This time I am sure my plasmid has been completely linerized. I will follow your suggestion and run a gel after ligation.

Is there a possibility that a vector without compatible ends will get self-ligated?

#9 kant0008

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Posted 22 September 2004 - 04:23 PM

Unless there is somethign reaaly weird going on in your system, no , it shoudln't:)

#10 postdoc

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Posted 23 September 2004 - 12:58 AM

So then if you run that alongside your annealed product it should run differently if your 100bp fragment is ligated, as ligated plasmid should run higher (normally) than cut.


Another question, Is that possible comparing the size of unligated hence linear vectors vs ligated hence circular vectors, especially there is only 100 bp difference in size? If yes, what percentage of gel would you suggest?

#11 kant0008

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Posted 23 September 2004 - 09:23 PM

Linear, unligated plasmid will run at the molecular weight, will correspond to marker. Ligated, closed plasmid will run "anywhere", normally somewhere higher than the linear form. So if you mean compare size by base pairs, no it's not possible. If you mean compare size to determine whether the plasmid closed- then yes, it's possible.
The gel percentage- depends on the size of your plasmid, the bigger the plasmid the less concentrated the gel- to have good resolution about the right area. Start with 1%.
Good luck!




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