T4 Ligation Failing - (Aug/11/2012 )

Hi All,

My first post here on the PO forums. I'd like to thank everyone for providing such an incredible resource that I frequent.

So I've been having some weird issues with a failed ligation for cloning. I have a purified vector and insert that came from digestion with EcoRI and HindIII. I know those pieces are good as I've run both on agarose gels (the vector is ~4000bp, and the insert is ~600bp). I've double checked the restriction sites, etc.

However, I have been trying to ligate these two into a plasmid for bacterial transformation using T4 DNA Ligase and T4 Ligase Buffer with ATP (both from BioLabs), and something weird is taking place in the process.

I am running this ligation at room temperature for an hour with 100+ ng vector/insert.

I believe that ligation reactions are, in theory, relatively inefficient, though I would expect that the ligation product be somewhere around 4600bp. Is that correct?

However, looking at the gel, it seems that there's no more vector, no more insert, and no product at 4600.

I've attached a picture of a gel containing the ligation product. The ladder you see is a 1 KB Plus Latter (Invitrogen).

Anyone have any idea as to what I'm looking at on that gel? I honestly don't have a clue. Thank you in advance.

I never look at ligation product on a gel, except for very rare diagnostic reasons. Just transform your ligation and check the resulting colonies.

phage434 on Sun Aug 12 01:16:52 2012 said:

That's what happened here. No joy on the transformation, so I ran the rest of my ligation product on the gel. Any idea on what the gel may indicate went wrong?

The general rule is that you want to keep the ligations below about 100 ng total DNA, and try a few different molar ratios of backbone: insert. I typically do 20 ng of backbone with however much insert for the different ratios.

Check that the ATP hasn't gone off in the ligation buffer - you can supplement with fresh ATP if needed.

OK. In general, the problem is not with the ligation, but either with the DNA going in, or with the competent cells. So, some questions:

1) Do you primers have 5' extensions after the restriction sites, or are you cutting DNA from another source rather than PCR?
2) How are you purifying your DNA?
3) How are you cutting your DNA? Volume? Volume of DNA solution going in? Enzymes?
4) How are you purifying your DNA (if at all) after cutting?
5) How are you quantifying your DNA prior to ligation?
6) What are your competent cells, and how were they obtained?
7) What is the measured competence of your competent cells? (cfu/ug of DNA)
8) What are your outgrowth conditions?
9) Are your plates good (correct antibiotic?) (Have you tested with controls?)

bob1 on Sun Aug 12 04:32:00 2012 said:

Bob -- thank you! I'll try that on Monday and report back. I will make a fresh stock of Lig. Buffer, and I never thought of trying a few different molar ratios for a set of reactions.

Do you then transform all of those, see what worked, and go from there?

phage434 on Sun Aug 12 12:07:54 2012 said:

Thanks for the response

1) Yes, from PCR, and there is something like GTG GTG....etc. after the restriction site.
2) Purification with gel extraction
3) Cutting with EcoRI and HindIII, 37 degrees for one hour, don't recall the exact volume of DNA going in.
4) Purification with Gel Extraction (vector) and PCR purification kit (insert)
5) Quantification of concentration with spectrophotometry
6) Used competent dh5a (someone else used the same ones and had success)
7) Have not measured competence - should I do that?
8) Outgrowth at 37 degrees overnight
9) Plates are good, and checked with + Control of the same original plasmid.

Anything there sound weird that may have thrown it off?

1) What do you mean "after"? Are they 5' or 3'?
2) Make sure you are not exposing fragments to too much UV during this step. I would eliminate it if you have clean PCR bands, and just do a pcr cleanup.
3) When most of the volume is DNA, you can inhibit this reaction with impurities from the DNA "purification". Aim for less than 25% of the volume being your input DNA.
4) This is probably not necessary since you can heat kill your enzymes. (always choose ones you can!). You may need to cut the band from your vector, but again be mindful of UV exposure. Try to minimize and use 365 nm not 302 nm UV (or blue light).
5) This is not a good way. Run a gel if you can with the DNA going into the ligation and compare band intensities. Specs can easily fool you.
7) Definitely if you are having problems. Dilute a common plasmid (pUC19 e.g.) to the 50 pg/ul or so level and transform with 1 ul, then count colonies. Back calculate the cfu/ug. Should have at least 10^8 or so for good competent cells. This is the most common problem.
8) Outgrowth in nonselective medium should be only for 1-1.5 hours. Overnight will select against your transformants, because the non-transformed cells will grow faster.
9) Good.

Thanks so much Phage.

For #1, the extension is at 5'.

I appreciate all the other advice. I'll keep trying next week with some changes and report back when I get it.

Thanks again!