Troubleshooting

Yesterday I kinased my SpecR PCR fragment, ligated it to the inverse-PCR fragment and transformed this into E. coli DH5alpha.   But my transformations gave only the same tiny colonies as the negative controls (no-DNA and no-ligation).  One of the positive controls (another AmpR SpecR plasmid made by the undergrad) gave thousands of AmpR and SpecR transformants, so I know that the competent cells (RbCl2-competent, frozen many years ago) and the antibiotic plates are fine.

(The other positive control was her pGEM-Spec construct; this gave no transformants for either Amp or Spec.  Since this prep didn't work as a PCR template either I should now throw it out.)

I wasn't totally surprised, because I already suspected that there was something wrong with the PCR amplification of the spec cassette.  The PCR product looked right when I ran an aliquot of the PCR reaction in a gel, with a sharp band just smaller than 2 kb.  But there was also a faint smear of smaller DNAs below this band, and after I had used a spin column to clean up the pCR reaction (should remove primers, enzyme and salts) the sharp band was smaller and blurry, and the DNA smear had become as second blurry band. The column cleanup of the inverse-PCR fragment gave a nice sharp band of the right size, so I don't think the problem was with the column treatment.

This was cause for concern, so I repeated the spec PCR, thinking that maybe I had screwed up (why would a cleaner band be smaller and blurrier?).  But I got the same result.  I again used the student's primers and template chromosomal DNA, but I lowered the annealing temperature for the first two cycles because the 5' 12 bp of each primer are not complementary to the template DNA. Before cleanup the amplification product looked the same as before (red arrow in left gel) - a sharp band with a faint smear below it (the smear doesn't show up in the photo).  After cleanup it again turned into a slightly smaller blurry band with a second blurry band below it (two red arrows in the right gel).  The larger sharp band indicated by the white arrow is the inverse-PCR fragment after identical cleanup.

I have no idea what could cause the SpecR PCR fragment to behave like this.  Before I dig into it I should check that the cloning failure was not instead caused by a failure of the kinase or ligation reactions.

Test for kinase and ligation:  I'm taking the inverse-PCR fragment, and kinasing and ligating it.   This should produce a simple plasmid with the antitoxin gene deleted.  I'll transform this into DH5alpha, selecting for AmpR. (Again no-DNA control and p∆AT:spec control.)  If I get lots of transformants then the problem is the Spec PCR fragment.  If I get none then the problem is the kinase or ligase reaction.  I was hoping to use singly-cut p∆AT:spec as a ligation control, but neither SacII nor HincII appear to cut where they should, so I'm doing without this control.

Just doing it

My inverse PCR reaction using the Q5 High Fidelity polymerase (creates blunt ends) worked on the first try!  I now have the antitoxin-deletion fragment I can ligate to a SpecR cassette to create the antitoxin knockout plasmid. Running 1/10 of the reaction in a gel gave a reasonable band, so I think I have enough DNA for the next steps.

I was initially planning to cut the SpecR cassette out of a plasmid the Honours student made, but we didn't have any of the blunt-cutter (BstZ1) this would need.  Since money is tight, I decided to instead do what the Honours student had done, using her primers to amplify the fragment with the Q5 High Fidelity polymerase, adding 5' phosphates with T4 polynucleotide kinase and ATP, and then blunt-end ligating this with the inverse PCR fragment.

The SpecR PCR didn't work the first time, using the Honours student's plasmid as template.  The plasmid DNA didn't look very good in a gel, so after consulting with the Honours student by email I tried using some chromosomal DNA she had made from another knockout mutant.  This worked very well.  My only concern is that in a gel the SpecR fragment had a short smear of what looked like shorter DNA below it (sorry, forgot to save photo image).

Next step will be to clean up both PCR products.  The Honours student did this by gel purification, using a kit favoured by the sabbatical visitor, but I think I'll just use our usual spin columns.  The grad student warns me that the recovery won't be great, but I think I have DNA to spare.

Next step will be to phosphorylate the 5' ends of the SpecR fragment.  This procedure looks very straightforward.  I could also treat the inverse PCR fragment, but this would allow that fragment to ligate to itself, creating many side reactions that I don't want.

Then maybe another spin-column step, because I think I should remove the kinase so it doesn't phosphorylate the other fragment. But I could instead just heat-inactivate the kinase (20 mi at 65°C, says NEB), since the kinase and ligase enzymes have nearly identical reaction buffers, and both use ATP.  After heat-inactivation I'd just need to add the other fragment, the ligase, and maybe a bit more ATP.

Then transform into competent E. coli (IDH5-alpha? I'm pretty sure I have lots in the freezer) and plate on spectinomycin.  The plasmid I want should give resistance to ampicillin too.

What about controls?  (I'll only do the easy ones this time.  If the transformation fails I'll do more controls.)

• Negative control: Mix of fragments before addition of ligase.  
• Negative control for transformation: No DNA.  
• Positive control for ligation:  I'd need to digest a plasmid.
• Positive control for kinase:  Do the ligation reaction with non-phosphorylated DNA
• Positive control for transformation: One of the Honours student's successful plasmids.

Fear of cloning: time to stop stalling and just do it!

For several months I've been stalling on a relatively simple project whose completion would let us submit a nice paper.  This is the missing step in an undergraduate Honours student's project; I wrote about her project and what I need to do here.

1. Starting with a circular plasmid containing the short 'Toxin' and 'Antitoxin' genes, I'll use inverse PCR to amplify a linear fragment that lacks most of the coding sequences of the Antitoxin gene.
2. I'll also amplify (or find) a SpcR cassette.
3. I'll ligate these two molecules together to create a circular plasmid with the SpcR cassette replacing the Antitoxin gene.  I could do this by blunt-end ligation (what the Honours student originally did) or do it as the student originally planned, using conventional ligation of 'sticky ends' generated by digesting both fragments with a restriction enzyme whose site is present at all the ends (she designed it into the primers).  I think she changed her plan because our stock of this enzyme was inactive.
4. I'll use this plasmid (linearized by cutting somewhere in the vector) to transform the bacterium Actinobacillus pleuropneumoniae to SpcR.
5. I'll use PCR of chromosomal DNA from the new resistant transformant to check that the original antitoxin gene has been replaced by the SpcR cassette.
6. Next I'll do a transformation assay on this mutant to see if its competence has changed, with the wildtype and toxin mutant as positive controls.

Step 1 actually requires that I get off my butt and:

1. Resuspend the primers at the appropriate concentration of TE (or water?).  (Check with the grad student.)
2. Find the PCR reagents. (Ask the grad student).
3. Find the template.  (Find info provided by Honours student before she left.)
4. Learn how to run the PCR machine (Ask the grad student.)