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3 way ligation help needed


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#1 Stuck with Ligation

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Posted 13 November 2009 - 12:13 PM

I have 3 pieces of DNA that I need to ligate. They are all ~700 bp. The first piece has the start of gateway cloning site at the 5' end and the 3' end has a NotI site. The second piece has an NotI site at the 5' end and an XhoI site at the 3' end. The 3rd piece has an XhoI site at the 5' end and the start of the end of the gateway cloning site at the 3' end.

When I try to ligate the three pieces together I see a ladder on the gel with very few bands at the ~1800bp site. I have tried band stab PCR and have had no luck amplifying these bands (they could just be 3 random pieces ligated together). I have tried my ligations at RT for 10-30 min and the ligation always produces a ladder on the gel.

When I try to ligate only 2 of the piece I am having the same issue where I see a ladder.

Does anyone have any advice?



I cannot put the pieces directly into E. Coli yet because I need to run a round of PCR on the correct band to finish putting on the gateway cloning site.

I have thought about getting the TOPO kit and screening colonies but we do not have multichannel pippetts and am afraid that this will take forever since I have hardly any bands of the correct size. I am debating running the ligations on a gel and excising the correct band size (even if nothing is really shown) and trying to transform hoping the small amount of DNA that is there could get into the TOPO vector so I can replicate it in the bugs and do a colony PCR to amplify my region.

#2 Warren

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Posted 14 November 2009 - 06:30 PM

dephosphorylate your second fragment (the one with the xhoI and notI sites)...

warren..

#3 Stuck with Ligation

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Posted 16 November 2009 - 12:35 PM

Would that leave a system where there are no phosphates?

NotI leave the phosphate on the 5' end of my cut first fragement would have:

5'-Seq-GC-hydroxyl-3'

and my middle (xhoI and Not I would have)

5'-phosphate-GGCC (notI)-sequence-XhoI seq-hydroxyl-3'

and if I use CIP then it would remove the 5' phosphate so I'd be attempting to ligate a 3' end that has a hydroxyl from the first piece to the CIP treated 2nd piece with the 5' hydroxyl removed. I am assuming this would lead to no ligation.

#4 Warren

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Posted 16 November 2009 - 12:49 PM

Would that leave a system where there are no phosphates?

NotI leave the phosphate on the 5' end of my cut first fragement would have:

5'-Seq-GC-hydroxyl-3'

and my middle (xhoI and Not I would have)

5'-phosphate-GGCC (notI)-sequence-XhoI seq-hydroxyl-3'

and if I use CIP then it would remove the 5' phosphate so I'd be attempting to ligate a 3' end that has a hydroxyl from the first piece to the CIP treated 2nd piece with the 5' hydroxyl removed. I am assuming this would lead to no ligation.


DNA is double-stranded, so for instance in your first example you are showing only one of the strands, the other strand (with the 5' overhang) WILL be phosphorylated:

5'-Seq-GC-OH-3'
3'-Seq-CGCCGG-phos-5'

If you just dephosphoryate the middle fragment, the three fragments will ligate together the way you want, with a couple of nicks -- I'd probably try to clone it first before doing PCR. If your Gateway sites are blunt ends they can be ligated as well, but the overhangs will ligate preferentially.

#5 Stuck with Ligation

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Posted 16 November 2009 - 01:52 PM

I have to do the PCR because we do not have any cloning kits available to do the intermediate step. If the PCR doesn't work I can buy one. Thanks, I will have to try CIP treating the middle fragment and seeing if it works. I wasn't thinking of the 2nd strand being able to ligate with nicks only on one strand.

#6 Stuck with Ligation

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Posted 16 November 2009 - 04:01 PM

I tried it and it didn't work. I am wondering if I let the CIP reaction go to long. I let it sit for 10 min to cut 150 ng of DNA and the phosphate was removed on the 3'-5' strand.

#7 Warren

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Posted 16 November 2009 - 09:39 PM

I tried it and it didn't work. I am wondering if I let the CIP reaction go to long. I let it sit for 10 min to cut 150 ng of DNA and the phosphate was removed on the 3'-5' strand.


ummm, ok, there is pretty much no way you could have done everything in the 2 hours since your last post, so I am wondering how you can know "it didn't work"? I guess I need to explain in more detail. I will operate under the assumption that your three fragments are PCR products (or at least the outside fragments, so your "Gateway sites" are not phosphorylated to begin with, ie they were part of the primer sequence of the original PCR). So, after the original PCRs, you do a PCR cleanup (ie column) or a gel purification. Then you digest your 3 fragments separately with the appropriate enzymes. Then you would take the middle fragment only, and dephosphorylate it. Then you would inactivate the phosphatase, and repurify your three digested fragments (to get rid of the small overhangs you cut off)...then add all three in equimolar amounts to a ligation reaction. In the ligation, everything that can happen will happen, so you will end up with bands at 700 (single fragments, may not be visible), 1400 (end fragments self-ligating) and 2100 (what you want, ie, the three correct fragments together). Nothing else should be possible if everything works (and your blunt gateway end can't ligate, ie, from a PCR). Then you would isolate the 2100 bp fragment from the gel and either clone it, or do PCR directly on it. You need to do this last purification as well, because if you don't, your 1400 bp fragments, which will be NotI or XhoI ends ligated together, will be able to amplify using just one of the outside primers, and would cause big problems in the PCR.

If you draw it out on a piece of paper, it might be easier to visualize. But it should work. The only problem would be doing PCR on nicked fragments, but there is enough overlap that you should get what you want.

warren..

#8 Stuck with Ligation

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Posted 17 November 2009 - 01:38 PM

I tried to ligate with the middle piece being CIP treated yesterday using (I had done all the other steps you suggested in the later post already and had the dna cut and cleaned up). I figured that since I only saw bands corresponding to the starting material that the ligase was bad so I acquired some new ligase and religated 100 ng of each of the 3 pieces (all similar size so 1:1:1 molar ratio) at room temperature for 1 hour (I have heard that NotI likes room temp ligation due to the high GC overhang). After running the ligation on a gel. I see bands at 700 and 720ish (which are my starting materials for the 1st and 3rd pieces) and a band at ~1400 (for any of the two pieces ligated) but no bands at 2000 plus.

I am now thinking I might have to put one piece into a cloning vector and purify and then use this cloning vector to put in the 2nd piece and so on. I really don't want to since it is time consuming but this 3 way ligation is killing me. I may also try ligating 2 of the pieces together and then PCRing and then trying to ligate this fragment with the 3rd piece.

#9 Warren

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Posted 17 November 2009 - 07:30 PM

I tried to ligate with the middle piece being CIP treated yesterday using (I had done all the other steps you suggested in the later post already and had the dna cut and cleaned up). I figured that since I only saw bands corresponding to the starting material that the ligase was bad so I acquired some new ligase and religated 100 ng of each of the 3 pieces (all similar size so 1:1:1 molar ratio) at room temperature for 1 hour (I have heard that NotI likes room temp ligation due to the high GC overhang). After running the ligation on a gel. I see bands at 700 and 720ish (which are my starting materials for the 1st and 3rd pieces) and a band at ~1400 (for any of the two pieces ligated) but no bands at 2000 plus.

I am now thinking I might have to put one piece into a cloning vector and purify and then use this cloning vector to put in the 2nd piece and so on. I really don't want to since it is time consuming but this 3 way ligation is killing me. I may also try ligating 2 of the pieces together and then PCRing and then trying to ligate this fragment with the 3rd piece.


Piecing it out might be easier in the long run -- sometimes easier techniques that appear to be more work or take longer, actually end up working quicker, I know this from experience!

However, if you were getting ladders before and now with a dephosphorylated middle fragment you are only getting your starting product only and single ligations, that is strongly suggestive that one of your restriction sites is not being cut, and based on your results so far (if I am understanding them correctly) it would most likely be one of your outer fragments. If you have some left over, you could check this quickly (I am assuming these are PCR products and will not ligate unless cleaved) -- just do a couple quick ligations with each of your outer fragments alone, which if they are cut correctly should yield a strong "double" band of ~1400 and a smaller band of ~700. The problem here is if one of your enzymes isn't cutting, doing it a longer way as you have suggested will not be any easier! How close to the end are your XhoI and NotI sites?

Another thing you can also do, since you mentioned the strong GC overhang, is briefly warm the sample to say 37 or 42 (not enough to denature the DNA, but enough to separate 4bp overhangs temporarily) will all your fragments are together right before you add the ligase (cool it back to room temp before adding the ligase). I do this sometimes, I don't know how much it helps, but I know it doesn't hurt.

Warren..

#10 Stuck with Ligation

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Posted 18 November 2009 - 06:43 AM

The two bands did make me think that only one piece is ligating.

I am wondering if my NotI is not cutting. Each of my PCR products has an additional 6 BP's after the restriction site. The base pairs are TTT TTT (which has worked successfully for me in the past). I have done some reading that NotI likes 8 BP's but the NEB webpage recommenced 6 BP after a restriction site. I don't have any positive controls for NotI (since I picked it because it cuts only my fragment) but the enzyme is good (purchased a few months ago and used only a few times).

#11 Stuck with Ligation

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Posted 18 November 2009 - 07:22 AM

According to NEB:
Q14: Is there a difference in cutting close to the ends between NotI-HF and NotI?

A14: No. When tested on a series of five oligos (19-23 bases in length) containing the restriction site and an additional 1 to 5 A/T bases from the end, both enzymes cut the oligo with 1 base pair from the end. When designing primers, NEB recommends adding 6 extra bases to ensure against experimental failure due to variation in substrate and reaction conditions.

So the NotI should cut and the XhoI I have is an aliquot from another lab which claims they use that same tube regularly with no difficulties.




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