Ligation & Troubleshooting

Immediately Follows: Restriction Enzyme Digest Protocol

 
 

Equipment and Consumables:

Protocol:

Before you start; Read the following section about setting up the 4-5 Controls necessary to debug this experiment when it fails (ligations can be tricky!). These are necessary in order to determine whether a digest/ligation succeeded or failed. Using controls will allow you to immediately recognise a false positive, avoiding weeks of unnecessary work downstream. They will also allow help you accurately pinpoint which part of an experiment failed. Setting up controls = creating a few extra mixtures for the Heat Shock Protocol and using a few extra agar plates. It seems like a hassle, but it is worth it - trust me!
This ligation protocol is for your experimental tube, follow the instructions below to learn how to adapt this protocol for each Control.

  1. Retrieve the T4 ligase enzyme and buffer aliquot from the -20°C freezer and put them IMMEDIATELY on ice. These reagents are VERY heat sensitive, and must be handled with care, explained above.

  2. Put your labelled tube(s) on ice, then set up the ligase reaction in this tube on ice. Be careful not to get ice or melted ice in the tube - this is not sterile! Add reagents in this order, change tips each time :

    - 2 µl of 10 x ligase buffer

    - 8 µl purified insert DNA

    - 8 µl purified plasmid DNA

    - 2 µl of T4 DNA ligase enzyme

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    Total 20 µl

  3. Mix by flicking briefly then incubate either 4°C overnight or room temp for 1 hour. Return the ligase enzyme immediately to -20°C freezer. Throw out any unused thawed ligase buffer.

  4. Proceed to Heat Shock Protocol or Electroporation Protocol to insert your ligated plasmid into your host of choice.

  5. If your plates have colonies growing on them after the recommended incubation period, proceed to the Patch Plate and Colony PCR Protocol to start the screening process.

  6. If things don’t work, see following protocol for details of troubleshooting ligations.

Summary of Digest and Ligation Controls:

1. Negative (no plasmid) – are the antibiotic plates OK? (expect no colonies)

2. Positive (uncut plasmid) – are plasmid stock + comp cells OK? (expect very many colonies)

3. Digest (cut plasmid) – are the restriction enzymes working? (expect very few colonies)

4. Ligation (cut, religated plasmid) -  is the ligation working? (expect many colonies)

5. Dephosphorylation (cut, dephos, religated plasmid) – is phosphatase working? (expect few colonies)

  • Only perform if you’ve done a dephosphorylation step (ie. Your reaction used only one restriction enzyme.)

Detailed Explanations of Digest and Ligation Controls:

  1. Transformation negative control: 50 µl of competent cells, no DNA added.

    • This control is primarily to check that you have made the antibiotic agar plates correctly, and it should yield no colonies at all.

    • If you see lots of colonies here, this most commonly means either you forgot to add antibiotic to the plates, or the antibiotic concentration is wrong (too low), or the host bacteria are already resistant to the antibiotic (e.g. TOP10 has chromosomal streptomycin resistance).

    • If you see just a few colonies on these plates, this indicates some kind of contamination has occurred during the procedure, e.g. from one of the other samples or the pipette etc. This may not be a ‘deal-breaker’ so long as there are lots more colonies on your experimental test plates.

    • Other possibilities could be;

      • The plates were incubated too long (especially with LB-ampicillin),

      • That there is severe contamination with an antibiotic-resistant  bacterium (not E.coli) (this is unlikely!),

      • That there was a mix up of labelling somewhere e.g. is this really the positive control?

  2. Positive control: 50 µl of competent cells + 1 µl of conc. plasmid

    • This control is to check that your stock of the plasmid vector is OK, and that your competent cells are indeed competent; it should yield thousands of colonies on the ‘pellet’ plate.

    • If you see no growth here, or only a handful of colonies, possibilities are as follows:

      • The cells are not competent

      • Used the wrong antibiotic in the agar (check the sequence of your plasmid to confirm correct resistance)

      • Used the wrong concentration of antibiotic (too much)

      • The agar plates are ‘bad’ for some other reason (e.g. added mercuric chloride instead of sodium chloride!)

      • The plasmid stock has gone bad (run a gel to check)

      • There was a mix up of labelling

      • There was a pipetting error (look at the pipette tip to ensure you really have 1 µl of plasmid in there!) 

  3. Digest controls. 50 µl of competent cells + 3 µl of purified digested plasmid

    • This control is to check that your restriction enzymes are cutting the plasmid vector effectively. It should yield only a few colonies (approx. < 20 on the ‘pellet’ plate).

    • Set up digests as described in the Restriction Enzyme Digest Protocol, as if you were going to ligate the plasmid to an insert (ie. 250 ng plasmid in 100 µl digest, then after incubation, purify on column, and elute in 15 µl EB), but don’t actually set up the ligations, just transform the purified, digested plasmid directly into the cells.

    • The digest controls should be interpreted alongside an agarose gel run with the remainder of the digested, purified plasmid; the latter should give a single sharp band at the expected total size of the vector plasmid. If you see a lot of smearing in this digest this could indicate non-specific nucleases are contaminating the reaction. If you see additional bands in addition to the band at the expected size, this could indicate incomplete digest (this is quite common) – these extra bands are the supercoiled circular and/or open-circular forms of the plasmid – you need to remove these by re-doing the digest with less DNA and/or more-purified DNA and/or a larger volume digest. The uncut plasmid bands tend to look ‘fuzzier’ than the cut plasmid.

    • If you have undigested plasmid remaining in the mix, you will get a very high background of vector-only clones, which will make it hard to find your clones of interest. The digest controls will also reveal if your chosen restriction enzymes definitely cut in the expected locations, or if there are other unexpected cut sites in the vector backbone (bad!) – that will give multiple sharp bands, which add up to the expected total plasmid size. 

    • If you see hundreds or thousands of colonies here;

      • The restriction enzyme has gone bad

      • The digest was set up incorrectly

      • Your plasmid stock is not sufficiently pure

      • You have put too much plasmid into the digest.

      • A separate digest control is needed for each enzyme you are using for cloning – e.g. if you are cloning an EcoRI-XbaI fragment into a vector cut with the same enzymes, you need to test EcoRI digestion and XbaI digestion separately.

  4. Ligation control: 50 µl of competent cells + 3 µl of purified, single-digested, religated plasmid

    • This control is to check if the ligation step is working. It should yield hundreds of colonies on the ‘pellet’ plate.

    • Set up digests and ligations as described for the standard ligation procedure, but using a single restriction enzyme only, and no phosphatase step, and no insert DNA.

      • If you are cloning with two different restriction enzymes, you need to prepare two separate ligation controls, since a double-digest would not be expected to religate in the absence of insert DNA. 

    • This control needs to be interpreted side-by-side with the other controls listed above to ensure that the problem is not poor-quality plasmid DNA or non-competent cells or bad agar plates etc.

    • If you see only a few colonies or no colonies, this most likely means that either the ligase enzyme is bad or the ligase buffer is bad. It could also be due to:

      • Bad ligation setup or conditions (e.g. a bad batch of MQ water).

      • There are non-specific nucleases getting into your restriction digest – this would mess up the ends of the DNA and prevent it religating. (check digests on gel; they will look smeary if you have non-specific nucleases).  

        • You may need to improve your sterile technique and sterilisation procedures.

        • Invest in gloves

  5. Dephosphorylation control. 50 µl of competent cells + 3 µl of purified, single-digested, dephosphorylated, religated plasmid. Only do this Control if you needed to dephosphorylate.

    • This control is to check that the phosphatase enzyme is working. It should yield very few colonies (approx <20 on the pellet plate).

    • Set up the digest, dephosphorylation, and ligation as described for the standard ligation procedure, but using a single restriction enzyme only, and no insert DNA.  In this situation, the ligase will attempt to join the cut plasmid backbone to itself, but it should fail to do this since the 5’ phosphate groups have been removed. This control must be interpreted alongside the other controls to rule out e.g. lack of restriction digestion if you see many colonies appearing.

    • If you see hundreds or thousands of colonies, it means that the phosphatase enzyme or buffer is bad, or you forgot to add phosphatase buffer or enzyme.

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