DAMN! Complete PCR failure!

Yesterday I ran a PCR amplification using DNAs from single colonies of 7 different A. pleuropneumonia isolates, and got absolutely no DNA fragments from any of them.

This amplification worked fine last time.  Can I figure out what went wrong?

• I checked the run record of the PCR machine - it looks fine.
• I checked the freezer box with the tubes of dNTP stock, 5X buffer, and Q5 polymerase, to be sure I hadn't picked up a wrong tube.
• I checked my notes, to be sure I hadn't left out any component of the reaction mix.  I'd checked off each reagent as I added it, and the final volume was as expected.
• I checked the 'F' and 'R' primer tubes (in another freezer box) to make sure I'd used the correct ones.  I'd made up more of the 10 mM dilution stock, so I also checked that I'd used the right tubes of the more-concentrated 100 mM stock to do this.  I even checked the remaining volumes in the two primer tubes - if I'd added one primer twice and not the other these volumes should differ by about 17 µl, but they're within a few µl.
• I prepped the colony DNAs slightly differently.  Last time (prep 1) I put a whole colony into 100 µl of medium, then diluted 5 µl of that into 45 µl water and heated to 98 °C for 10 min to lyse the cells and free their DNA.  This time (prep 2) I put part of a colony into 100 µl water, heated that, and then pelleted out any cell debris.  Both times 1 used 1 µl of the heated sample.

What could I try now?

• Use leftover Prep 1 colony DNA as template
• Vortex the Prep 2 colony DNA tubes
• Use as template purified DNA from lab stocks
• Use a different pair of primers (the Spec-cassette ones worked well last time)
• Repeat with the same reagents and template I used this time
• Make fresh colony DNA preps
• Make proper DNA stocks to use as templates

Plan:  

• Prep 2 14-1 colony DNA, Spec primers
• Vortexed Prep 2 14-1 colony DNA, F & R primers
• Prep 1 14-1 colony DNA, F & R primers
• Prep 1 14-1 colony DNA, Spec primers
• 1/100 dilution of lab-stock DNA, F & R primers

Success

When I last posted, nearly 3 weeks ago, my first attempt to generate the desired full-length knockout construct had given a mixture of fragments rather than just the desired full-length one.  But this mixture did include a relatively faint fragment of the desired size (3.6 kb).

I did try to get a better PCR product, but increasing the annealing temperature made things worse, and I couldn't find a PCR app that would let me diagnose which incorrect-priming reactions were producing the unwanted fragments.  So I went ahead and transformed competent Actinobacillus pleuropneumoniae cells with the mixture, selecting for spectinomycin resistance.

My logic was that only the desired fragment is likely to efficiently transform cells to SpecR, because other fragments were unlikely to have the correct homologous DNAs flanking the SpecR cassette.  If the 3.6 kb fragment was what I hoped it was, I should get thousands of transformants even though it was only about 10% of the total DNA in the mixture.  If it wasn't what I wanted, then it would probably transform very inefficiently if at all and I would get very few transformants.

I got thousands of transformants in my first try.  Since the real goal of this project is to find out whether knocking out the antitoxin gene prevents transformation in A. pleuropneumoniae as it does in H. influenzae, I did a quick-and-dirty competence assay, using 7 pooled SpecR colonies and some kanamycin-resistant A. pleuropneumoniae chromosomal DNA.  This gave lots of KanR transformants, but luckily I didn't take this as a final result.

Instead I went back and redid the transformation of A. pleuropneumoniae with the PCR mixture, this time using a lot less  DNA.  I did this because the high DNA concentration used in the original transformation meant that many cells could have taken up multiple DNA fragments.  In H. influenzae such fragments are known to undergo ligation in the periplasm, allowing formation of chimeric recombinants that give very confusing results.  Using 100-fold less DNA still gave plenty of SpecR transformants, and I streaked 4 of these to get clean single colonies.  (Two of the picked colonies were large, and two were smaller, but all gave large colonies on their streak plates.)

I tested 2 of these colonies by PCR.  Only one (14-1) gave the expected 3.6 kb full-length product and 1.1 kb Spec cassette products.  The other (14-2) gave no product with the full-length primers and what looked to be a slightly small product with the Spec primers.  The control wildtype cells gave the expected 2.6 kb full-length fragment and no Spec fragment.

At the same time I tested both colonies for the ability to be transformed.  Both were defective, with transformation frequencies 100-fold lower than the wildtype cells.  This is the most interesting result - it suggests that the Toxin-antitoxin system in A. pleuropneumoniae plays the same role in competence as its homologue does in H. influenzae.

Next steps: More comprehensive characterization of all the A. pleuropneumoniae mutants.  First do the full-length PCR on colonies 14-1, -2, -3 and -4, on the ∆toxin and ∆toxin/antitoxin mutants made by the honours student, and on the wildtype control, this time running the gel more slowly to better characterize the fragment lengths.  Then do additional PCRs using other primers, to confirm the mutant structures, and repeat the competence assays on all these strains.

I'll also need to get all the final mutants sequenced, to confirm that they have only the expected deletions. I'll email the former RA to ask her the best way to do this (do I send genomic DNA or PCR products, what primers are best...).

Semi-success

Here are the results of the first attempt at getting full-length PCR products:

In the left lane (high template) I see a faint full-length band, and a stronger band the expected size of one of the expected intermediates.  In the right lane (low template) I see only the intermediate band.

This is a fine result.  I'm now using 0.5 µl of the high-template reaction as the template in a new reaction with the same F and R primers and an annealing temperature optimized for them.  I hope this will give me lots of the full-length product.

In anticipation of having the desired full-length DNA fragment, I've just streaked out the recipient Actinobacillus pleuropneumoniae cells I will transform this fragment into.  There are several steps I need to do before the final transformation:

• Streak out the honours undergrad's A. pleuropneumoniae SpcR mutants (she made three different ones with the same cassette I'm using).
• Check the sensitivity of A. pleuropneumoniae to spectinomycin, since this is the selection I will be using for transformation by my fragment.  The honours undergrad did this but her notes are not very good here.  I need to identify a concentration that will prevent colony formation by the sensitive cells but allow it by the resistant cells.
• Make a competent stock of the recipient (SpcS) by growing the cells in MIV starvation medium.  
• Check the competence of these cells by transforming them with genetically marked DNA.  I know I have some old DNA for this purpose (NalR?), but it would be good to select for SpcR using DNA from one of the undergrad's SpcR strains, if I can find this.

Before doing the final transformation I should also digest my transforming fragment with a couple of diagnostic restriction enzymes, just to be sure it is what I want.

Progress! The big fragment-assembling PCR is running!

The PCR amplification of the SpcR fragment worked fine yesterday (after I spent an hour staring at (forward and reverse and complement and reverse-complement) primer sequences to reassure myself that I'd ordered the correct ones).  And today I did a column cleanup and a gel purification.  And now I'm running the PCR reaction that I hope will assemble the complete DNA fragment.

The part I'm doing now is shown below.  You can see the whole plan in this post.

When I was getting ready to do the PCR I realized that first I needed to think in more detail about the events.  So here they are, in point form:

1. Most of the green strands will just reanneal to their complements.  Probably many of the red and blue strands do too.  This is an unwanted process that reduces the availability of strands for the desired annealings and elongations. In normal PCR this is hindered by the very high concentrations of the primers, but here we only have the F and R primers.
2. Primer F anneals to the lower red strand and primes synthesis of the upper red strand.  At the same time, primer R anneals to the upper blue strand and primes synthesis of the lower blue strand.  Both of these new strands accumulate linearly, not exponentially.  This provides more of the appropriate strands red and blue for STEP 4.
3. STEP 4 above: Sometimes the upper red strand anneals to the lower green strand (by their 17 bp of complementarity), and elongation in both directions produces hybrid upper and lower (red + green) strands.  Similarly, sometimes the upper green strand anneals to the lower blue strand (by their 16 bp of complementarity), and elongation in both directions produces hybrid upper and lower (green +_ blue) strands.  All these strands also accumulate linearly, not exponentially.  They provide the effective strands for STEP 5.
4. STEP 5 above: Sometimes the hybrid strands from STEP 4 anneal to each other instead of to their complements (by their 1260 bp of complementarity). Elongation in both directions produces the full-length strands.
5. STEP 6 above: Now primers F and R can anneal to the full-length strands, leading to exponential amplification of this desired product. 

Miscellaneous thoughts:

• I don't know how much template is appropriate, so I'm trying two concentrations, one 100-fold lower than the other.
• The rate-limiting step is STEP 4, the annealing of the red and green strands by their 17 bp overlap, and of the green and blue strands by their 16 bp overlap.  I'm using a fairly low annealing temperature to facilitate this.  If I don't get any product I'll try lowering it further.
• If I hadn't mixed the red and blue fragments for purification I might have set up partial reactions (red + green and blue + green), to make it easier to monitor progress.  If the all-in-one reaction doesn't work I'll probably make some more of each fragment and purify them separately so I can troubleshoot in separate reactions.

Successful fragment cleanup

Last night I designed and ordered the two complex primers I'll need to amplify and insert the SpcR gene.  They won't get here until Monday, so today I did column and agarose-gel cleanups on the two other PCR fragments I'll be using

First I pooled what was left of the two PCR reactions (A and B).  The volume of each was about equal to the amount I had run in my gel yesterday (left panel below).  Then I did a column cleanup to get rid fo the bulk of the PPCR primers, using an old EconoSpin column that I'd revived by passing 0.2 N NaOH through it.

Then I ran all of the eluate from that column in one lane of a 1.2% agarose gel.  I used only about 1/5 of the usual concentration of Ethidium Bromide, and I viewed the gel only with our hand-held long-wave UV lamp to avoid UV damage to the DNA.  The bands were faint but clear and I cut them out  together in one gel slice.

Then I used our new Zymoclean gel-recovery kit to dissolve the agarose and recover the DNA, and ran 3 µl of the resulting 24 µl in unused lanes of the same gel I used yesterday, to see how much DNA I had recovered.

The results look great.  The bands are sharp and bright and the right sizes, and the intensities suggest that I've recovered most and perhaps all of the DNA I began with.  So now this prep is in the fridge, waiting for the other piece of the construct.

In the lab! Doing PCR! Successfully!

I have primers for the A-R and A-F sites shown in the previous post, but they weren't designed to work with the F and R primers I also have (F with A-R, and R with A-F).  But I decided to go ahead and test them anyway before I order the new S-F and S-R primers I will need for amplifying the SpcR cassette, and for linking the other amplicons to it.

The New England Biolabs primer evaluation software recommends against PCR using a pair of primers whose Tms differ by more than 5 °C, but I don't have much to lose, so I'm running them anyway.  I'm also testing another version of the A-F primer, designed by the honours student.

I'm using our high-fidelity Q5 polymerase instead of Taq, because I need the product to be error-free.  Unfortunately each pair of primers requires a different annealing temperature, so I'm doing three PCR runs, each with a single tube.

And tomorrow morning I can run a gel to see if any of them worked.

It's tomorrow, and OMG!!!, all three PCRs gave excellent products!

So I just need to design and order SpcR primers with tails that will base-pair to the inner ends of products A and B.

New no-cloning plan for the antitoxin knockout

The Methods section of a manuscript I was reviewing reminded me that it's possible to use PCR to create a gene knockout, and then transform the PCR product directly into naturally competent cells, without an intermediate cloning step.

We actually had tried out this method bout 20 years ago, when the H. influenzae genome sequence first became available, but after some experiments we decided that cloning was usually more reliable.  But now I'm in a situation where cloning is being very unreliable indeed, enough so that I can't bring myself to try again.  So instead I'm going to try the direct-transformation method.

Basic plan:

1. In two independent PCR reactions, amplify the left and right genome segments flanking the gene to be deleted (the antitoxin gene).  These segments need to be long enough to later allow efficient homologous recombination of the final construct into the chromosome. 
2. Separately amplify a Spectinomycin-resistance cassette (SpcR), using primers designed with tails complementary to the inner ends of the two genome fragments.  
3. Remove all primers from these PCR products and mix them together in a PCR reaction mix with no added primers.
4. Do one cycle of strand melting, strand annealing (of the primer tails), and strand extension by Taq. This produces two mid-length fragments that both contain the SpcR segment.
5. Do another cycle of strand melting, strand annealing (this time of the full SpcR segment), and strand extension by Taq. This produces one full-length fragment containing both genome segments and the SpcR segment.
6. Add the outermost genome primers (left primer of the left segment and right primer of the right segment) and carry out a normal PCR amplification.
7. Transform the resulting fragment into competent A. pleuropneumoniae cells.
8. Select for SpcR and confirm the new genotype by PCR.

Now I need to dig into the sequence file and primer files to figure out whether I can reuse primers we already have, and to design the new SF and SR primers with the appropriate tails.