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Help with 3-way ligation - and calculating molar ratios for 3-way ligations... (Feb/24/2009 )

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phage434 on Feb 27 2009, 07:02 PM said:

I would phosphorylate the annealed fragment (or order the oligos phosphorylated). In principle the ligation might work without this (repair happens in the cell), but I think you are asking for trouble relying on that.

sorry for delay...
Yes thanks for the tip, the smallest insert was made from annealed oligos and they were ordered with 5'phosphates on them.


So just an update on the 3-way ligation for perneseblue et al...
I didn't get any colonies at all after trying the 3-way with your suggested ratios. So I went to plan B which was to cut more vector as you suggested. It's taken so long because I had some problems with some old LB, and also realized that the plasmid is a low copy one, so I'm having to optimize the minipreps for this to increase the yield. I'll try again with more vector once I have it. And working on another experiment in parallel now..

perneseblue on Feb 26 2009, 02:45 PM said:

PhilS on Feb 26 2009, 12:27 PM said:

Many thanks perneseblue,
I will let you know how it goes. btw, why do you think I need to do colony PCR on 96 colonies, that seems like it might be overkill to me, as there -should- be only one way for the products to ligate together no? I know that you are worried about multiple inserts of the small 58bp piece, but is a gel going to be able to differentiate between 58bp? If you think yes then do you suggest doing the colony PCR's and then looking for the smallest of a group of bands that look about the right size and sequencing that one?

96... well... I use a multichannel pipette. Checking 12 colonies isn't much different from checking 96. It is a very handy thing to have. However if one did not have a multichannel pipette, the number would have be scaled down.

While there should only be one structure... this is biology. The ligase appears to be able to ligate complementary ends that have skipped a few bp (creating a lip).

As for the colony PCR, it is important that you amplify across the junction between insert and vector. Or in this case between two different inserts. PCR is sensitive enough to amplify left over DNA (from the ligation reaction) on the plate. While you are conduction a 3 way ligation, you only need to check one junction. If the junction exist, very often the rest of the plasmid is correct.

Thus I suggest that colony PCR be used, which amplifies across the junction between insert and vector. Should a positive colonies be detected by PCR. These colonies (at least 2) should be grown up and checked by other methods such as restriction digest and sequencing. You need to check at least two (I grow up 3) for the rare situation that one plasmid is structurally wrong or contains mutations.

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