Field of Science

So long since I've posted...

But here's an update on the RNA-seq saga.

We've been struggling to get evidence that our RNA preps are of good enough quality for library preparation and sequencing.  Because the Ribo-zero kits that we use to remove the rRNA from the samples are so expensive, we wanted to be sure the final mRNA concentrations and integrity would be good enough to make the equally expensive sequencing worthwhile.

Unfortunately my attempts at using a Bioanalyzer have been very frustrating (and it's not cheap - several $ per sample), with odd smeary/spiky patterns and poor recognition of the size standards. This is partly because the concentrations are so low and the 'pico-RNA kits are very fussy, and partly because it's just a fussy procedure and easy (for me) to make mistakes.

We sought advice from the person who will be preparing the sequencing libraries for us.  She said that samples that appeared bad on Bioanalyzer results often gave excellent sequencing results, and suggested that we have her prep an initial 8 libraries.  She would then pass these on to the person who does the sequencing, who could just run her initial quality checks on the libraries and tell us whether there were suitable for sequencing.  So that's what we did, and the news was good - these initial libraries look OK.

Now it's time to do the Ribo-zero and final clean-up on the rest of the samples, so they can be handed over for eventual library prep and sequencing.  There's a two-month backlog so we likely will get the results as a Christmas present.

Our sample processing had been a bit scattershot, so now I needed to go through the sample information and make sure we have the right samples, and that the numbers add up.  They do, so I think we're all set to quickly complete the remaining Ribo-zero treatments and use RNeasy-Min-elute columns to clean up each RNA sample after Ribo-zero.  Then the remaining 64 samples can be handed over.  (The 'we' here is our excellent UBC-Co-op program technician.)


We have accidentally wasted two of our Ribo-zero treatments on duplicate samples, and I was thinking that this meant that we'd have to buy another 6-sample kit ($650).  But now I think we can get by without this, either by not treating two samples (the G6 samples in the table above) or by using slightly less of the reagent for a subset of samples.

We had been using only 1 µg of total RNA for Ribo-zero treatment, but for these samples we'll sue all the DNase-treated RNA we have (typically several µg).  In some cases the volume of sample will be too high (max 28 µl for Ribo-zero), but singe the samples are in H2O we can just evaporate them a bit to decrease the volume.

Possible work with the new rpoD mutation

I'm up to my ears preparing materials for the new version of Useful Genetics, but we have a great new technician through UBC's Science Co-op program.  She's nearly finished the work preparing the RNA samples for our big RNA-seq project, so it's time to consider what she should work on next.

One possibility is characterizing the newly identified rpoD mutant strain.  This strain (RR753) has a point mutation in HI0533, which encodes the sigma factor that regulates initiation and early elongation of transcription of 'housekeeping' genes, especially during exponential growth.



One analysis we need is BioScreen growth curves of the mutant and a wildtype control, to confirm the preliminary growth curve data suggesting that this mutation causes slightly slower cell growth (lower graph).  I think this slower growth results from a general slowing or minor disruption to normal transcription of many genes, and is not specific to its effects on competence. 

My hypothesis is that the mutation's effect on transcription of sxy mRNA increases competence by increasing sxy translation.   I've long hypothesized that slowing elongation or increasing pausing in the 100 nt segment of sxy mRNA that forms its regulatory secondary structure will promote sxy translation by increasing the ribosome's access to the sxy ribosome-binding site and start codon.  We're not in a position to dive into the molecular analyses of RNA and protein that will probably be needed, but I wonder if there are some genetic or culture-conditions approaches that will shed light on the situation.
  • Is RR753 sensitive to the inhibition of competence by added purines?
  • What's the effect of an hfq deletion in this background?
  • How does this strain respond to added cAMP?
  • How does it respond to the standard competence-inducing MIV treatment?
  • Does the mutation increase competence of a sxy mutant (sxy6) that has an extra-stable secondary structure?
  • Does it further increase log-phase competence of the sxy hypercompetence mutants, which have weakened sxy mRNA secondary structures?
I think the technician is going to be doing a lot of competence time-courses...

CIHR results...so depressing

The results have been released for the latest competition for operating grants from the Canadian Institutes for Health Research (Canada's equivalent of NIH).  Our proposal was ranked 13/72 by its assessment committee (Microbiology and Infectious Disease), but they only funded 11.


This is our 7th failed CIHR proposal in a row.  They all had good scores, and several, like this one, were very close to being funded.  The scores show that our proposals do keep getting better (this time 4.5/5), but the funding cutoffs also keep rising and we're never quite good enough.

Will I try again?  Perhaps not.  This was the last round under the old funding system, and the new system is even less favourable to the small-lab fundamental research that we do. 

I'm not quitting research.  For this year we have funds left from a previous CIHR grant (must be all spent by the end of March), and after that we'll potter along on our very small NSERC grant.  Luckily most of what we do isn't very expensive, but I can now only support one grad student, supplemented with several excellent undergrads who'll work mostly for free.

More cell preps for RNA-seq analyses

Unfortunately we have to repeat most of the cell preps I made for the big planned RNA-seq analysis , because our RNA preps used up all the cells without producing any RNA.

The main problem was that we were using a Qiagen RNeasy Plus kit (designed to remove contaminating DNA) instead of the usual plain RNeasy kit.  I had bought the Plus kit because of a cheap introductory price, planning to just leave out the final 'on-column DNase digestion step, since this hadn't been very effective in the past.  However Qiagen had changed the kit without changing its name, replacing the final 'on-column' digestion with an initial pass through a 'G-DNA eliminator column' (before the usual RNeasy column steps).


The kit instructions didn't say anything about its suitability for bacterial cells, so I contacted Qiagen technical support.  They assured me that, although the kit would not remove DNA from bacterial cells, it would give a normal recovery of RNA.  What they didn't tell me was that the initial detergent treatment would not lyse bacterial cells - at least I think this must be the reason we didn't find any RNA when we ran all our RNA samples in a gel and a Nanodrop spec.  We had also processed some samples with the normal RNeasy kit, but most of these can't be used because we need complete sets of samples from replicate cultures.

Qiagen responded bvery well to my complaint about our results, providing us with two new RNeasy kits and 500 ml of the RNA Protect solution.  But we still have to do the work of regenerating the samples.  Luckily we have a very competent new technician, hired through UBC's Biology CO-op program, and she's doing most of the work.  In particular she's regenerating the samples of cells during induction of competence by our MIV starvation medium.  But tomorrow I'm going to (finally) spend a day in the lab generating all the samples from cells growing in rich medium.  This is 9 cultures, with 3 samples from each.

I think I can do them all in one day, if I plan carefully.  So here's some planning:

I'm collecting cells at three different densities:  OD600 = 0.02, 0.6 and 1.0.  I freeze pellets from 2 ml of cells for RNA prep (3 tubes with 0.67 ml cells and 1.3 ml RNA-Protect) and 1 ml of cells with 0.25 ml 80% glycerol, for later testing of transformation frequency if needed.  The OD 0.02 cells need to be concentrated 10-fold before freezing so we'll get enough RNA from them, so I need to start with a larger-than-usual volume of cells - last time I used 75 ml.

I'll be starting with the OD=0.02 cells frozen in glycerol in the last preps, rather than from fresh cultures.  These cells are already in log phase; I'll just pellet them to remove the glycerol and resuspend them in the 75 ml sBHI.  The initial density will be about OD=0.003, but previous experiments suggest that not all these cells will be viable, so the cultures may take several hr to grow back to OD=0.02. Then I'll concentrate 40 ml by filtration, resuspend the cells in 4 ml, and freeze 2 ml for RNA and 1 ml for competence assay.  The remaining culture (~ 20 ml after OD sampling) will continue shaking to OD=0.6 and OD=1.

I'll need to have the filters ready, and all the tubes to put the samples into, each labelled and preloaded with RNA-Protect or glycerol.

What are the cultures?

  • 3 replicates of KW20 (names on tubes: FK, GK, HK)
  • 2 replicates of murE749 (names: G7, H7)
  • 2 replicates of sxy1 (RR563; names: G5, H5)
  • 1 replicate of ∆crp (RR668; name: FC)
  • 1 replicate of ∆sxy (RR648; name: G6)

Why isn't competence regulated by the availability of DNA?


Most bacteria tightly regulate the genes that enable them to take up DNA from their surroundings.  This makes sense, since the uptake machinery is complicated, probably expensive to produce, and may interfere with other membrane functions, and since the benefits of DNA uptake may arise only under particular circumstances.

The regulatory signals include diverse physiological and environmental cues. In other posts I've discussed the signals that regulate H. influenzae competence, and here's a couple of recent reviews for Gram-negative and Gram-positive bacteria (http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6976.2012.00353.x/abstract; http://www.annualreviews.org/eprint/F2P5NJ3vsbWfbPGtusKN/full/10.1146/annurev.micro.60.080805.142139).  (Unfortunately neither is open access, sorry.)

But none of these bacteria are known to regulate competence by what should be the most important information - whether or not there's any DNA in their environment to take up.  I say 'known to' because in fact there is almost no published information addressing this point, and the researchers I've contacted don't know of any relevant data. 

It's possible that bacteria don't need this information because DNA is always within reach in their environments, but we don't really have data addressing this point either.

My lab has a new undergrad who doesn't yet have a project to work on.  I wonder if he'd like to test this point, first in H. influenzae and then maybe in other competent species?  Given the lack of positive evidence I'd expect negative results (external DNA doesn't affect competence), but I think this might be a case where negative results would be publishable.

The tests are tricky because addition of external DNA is also how we sensitively measure competence, so we might need a way to get rid of the 'inducing' DNA before measuring competence with the 'assaying' DNA. And good controls...

UBC's new website

Here's the email UBC sent everyone the day before they launched their new web presence:
"To the UBC Community,

We are pleased to announce that a significantly redesigned UBC.ca website will launch on April 25th, 2014.

Consultation and research showed that the current site could do a better job reflecting the true nature and scope of our university. The navigation, content
and functionality of the current site makes it difficult for our visitors to find the information they are looking for and the overall look is considered conservative and dated. The redesign addresses these challenges; it is bold and experiential, offering improved design, navigation and content. A few highlights of these changes are:

1. Moving from a solely internally-focused navigation structure to one that is also audience-based

2. Expanding the opportunity for faculties and units to share their content through a new homepage section called “UBC Now”

3. Creating innovative and in-depth stories on the homepage that illustrate the impact UBC is making in the world

4. Implementing secondary pages that provide a stronger introduction to internal UBC partner sites to improve site navigation and hand-off

We are encouraged by the positive feedback we have received from those who have seen the site while in development and will continue to monitor its performance and fine-tune as required following launch.

We wish to thank the large number of contributors from across our campuses for bringing their energy and ideas to the project.

We invite you to explore the site in the days ahead:

http://www.ubc.ca


Kari Grist
Managing Director
Communications & Marketing

UBC a place of mind"

 And here's the home page:



Actually there are several other versions with different photos, all of people evidently thrilled by what they're discovering at UBC.  The little dots in the ring around his head are links to pages about specific aspects of UBC's wonderfulness.

And here's the obligatory xkcd cartoon.  Rich FitzJohn found this and points out how obedient UC remains to its precepts.


A new mutation causing hypercompetence

I described last month how we were revisiting on old hypercompetent mutant whose causative gene was unknown.  I rechecked its phenotype and prepped DNA to sequence, from both the original EMS-induced mutant strain (strain RR735) and from a 'backcross' strain where the unknown mutation had been transferred to an unmutagenized genetic background by transformation (strain R753). 

Here's the phenotype again.  The lower graph shows that it grows slightly slower than wildtype, and the upper graph that it has a 10-200 times higher transformation frequency in the rich medium sBHI.


The postdoc just emailed me the sequencing results.  Both the original and backcross strains have the same single mutation, an amino acid substitution in the rpoD gene, which encodes the sigma-70 transcription factor.

This is a surprisingly clear result.  We had expected to find many EMS-induced mutations in the original strain, and probably several mutations in the segments of DNA transferred in the backcross transformation, and were planning another series of analyses to sort out which mutation causes the phenotype.  But both strains have only the one rpoD mutation, suggesting that our EMS mutagenesis wasn't nearly as heavy as we had thought.  As controls we had sequenced the original and backcross strains of another hypercompetence mutant (RR749, known to have a mutation in I, and both these strains also had only the single known mutation.

A hypercompetence mutation in rpoD fits very nicely into my thinking about how competence is regulated.  I'll write a separate post about this.

Cell preps for RNAseq are all done

I'm pretty sure that I've now done all of the cell preps for our big planned RNAseq analysis, more or less as diagrammed in the previous post. 

Instead of a cya knockout mutant as a negative control (pink in the diagram I used a crp knockout.  cya encodes the enzyme that synthesizes cyclic AMP (cAMP), and crp encodes the transcriptional activator CRP, whose ability to induce transcription is entirely dependent on cAMP, so the two mutants have the same phenotype - inability to induce both the competence genes and the energy-balance genes in the CRP regulon.  I decided to use the crp mutant partly because that strain grew up first and partly just in case there are traces of cAMP in our sBHI medium.  I did one MIV-competence time course with this mutant (4 samples) and one sBHI time course (3 samples rather than the 2 in the diagram).

I also did 3 sBHI-timecourse samples of the sxy knockout as another negative control (also pink in the diagram).  I think I now have two more samples than will fit in 3 lanes of sequencing (24 samples multiplexed per lane), so I'll probably omit the OD=0.6 samples of the negative control sBHI timecourses.  But I'll process the RNA from them just in case something goes wrong with another sample.

I found my missing DNase.  I hadn't lost it, just ordered the wrong kind.  So now I have $250 worth of a high-quality DNase I don't really need, and will need to order the right kind.

I'll get to the RNA preps once I get some teaching responsibilities under control - partly the grading for my face-to-face Human Ecology course but mainly the need to rerecord ~150 lecture videos for Useful Genetics/Genetics for Life.

RNA-seq progress, problems and plans

I've been growing the cell preps for the RNA-seq analysis, as shown in the planning figure below.

I did Day C's cultures, freezing 1 ml of cells for later transformation testing if needed, and 2 ml of cells for RNA purification.  The first snag was the invisibility of the cells!  Following instructions from the former RA, I mixed 2 ml cells from each of the first samples with 4 ml of the magic 'RNAprotect' reagent, let the mixture sit for 5 min at room temperature, and spun down the cells (2 ml of mixture in each of three 2 ml tubes) in our mini-spin microcentrifuge.  The plan was to discard the liquid and freeze the cell pellets at -80 °C, for later RNA preps.  But there was no visible cell pellet!  This amount of cells typically forms a small but easily visible pellet when centrifuged, but the bottoms of the plastic tubes looked perfectly clean.

I spun the tubes again - still no pellet.  So I pretended a pellet was present, discarded the liquid, and froze the apparently empty tubes anyway.  I checked the RNAprotect instruction booklet which reassuringly said that sometimes the pellet might be invisible, but I also contacted the RA, who said that her preps had given clearly visible pellets.  Later I thawed out one tube and did a wilful-suspension-of-disbelief RNA prep using the RNeasy kit, which produced the same concentration of (high-quality) RNA as the RA's original preps.  So I'm now assuming that there are invisible cell pellets in all the tubes, and I've done Day D's preps.

The RNAeasy kit also had some surprises, a solution that was supposed to be clear (or with a bit of particulates that could be removed by centrifugation), instead was very cloudy, and centrifuging it raised the cloudy material to the top (as a diffuse scum) rather than pelletting it.  I couldn't get rid of the scum (it just redispersed when I tried to pipette it), so I went on to the next step (adding 100% ethanol), which eliminated the cloudiness completely.  (Maybe this cloudiness came from the presence of too much residual RNAprotect in my tubes - because I couldn't see any pellet I didn't thoroughly drain the tubes before freezing them.)


I would have also tested the DNA-elimination step, which uses Turbo Dnase and a DNase-inactivator chemical.  But my brand new box of TurboDNase ($250) has gone missing.  I've searched the freezer a couple of times, and racked my brain in case I put it somewhere special for 'safekeeping', with no success. My next step is to go down to Stores and have them show me exactly what the TurboDNase box looks like, so I know I have the right search image.

I've also revised my plans for the cells I'll test.  As I wrote earlier, I'm only going to do one replicate of the ∆hfq mutant in MIV, since we really should use special precautions to avoid losing small RNAs from the prep (and maybe to do strand-specific sequencing).  Since we planned on three full lanes of Illumina sequencing, each 24-fold multiplexed, this change opens up space for 8 additional samples. Four of these will come from a MIV time-course using a crp or cya knockout strain (in Day E, which will be tomorrow).  This is an excellent control since it lets us identify all of transcripts dependent on the transcription factor CRP.  I'll also include both crp (or cya) and sxy knockout strains in the rich medium cultures on Day H; taking two time points for each will give the four additional cultures to complete the first two lanes of sequencing. 



Big prep of MAP7 DNA

We're almost out of the standard DNA that we use in our transformation assays.  It's chromosomal DNA of a strain called MAP7 (because it contains point mutations conferring resistance to seven different antibiotics).

So I grew up a liter of cells and prepped DNS from them.  Now I have 25 ml of nicely viscous DNA solution.  It's transparent and colourless but I suspect it's not really pure yet, so I'm doing a second purification on 0.5 ml just to check if the apparent concentration or purity changes when examined with the Nanodrop spectrophotometer.  I'll also do test transformations, with the last of the old DNA preps as a control.

Later:  The Nanodrop spec found that the repurified DNA had half the concentration of the big stock. So I ran a gel and found that the big stock still contains a lot of RNA.  My original RNase step must not have worked very well, probably because of the high concentration of SDS and of proteinase K.  So I';ve now incubated the prep with more RNase overnight.

Getting ready for RNA-seq cell/RNA preps

The RA's missing notebook hasn't turned up, so I don't have her notes of how she prepared the samples for the RNA-seq analysis. Luckily the main procedures are ones she used in many experiments and are described in her earlier notebooks and in an email she sent me.

The basic procedures:

Collecting samples:
  1. Grow cells to desired state in rich medium (sBHI) or competence medium (MIV).
  2. Mix 2 ml with 4 ml RNAprotect reagent (Qiagen); leave 5 min at RT.  We have 100 ml and can get more quickly through LSC Stores.
  3. Mix 2 x 1 ml with 0.25 ml 80% glycerol and freeze at -80°C. (for later competence assays).
  4. Pellet RNAprotect cells and freeze at -80°C.
Preparing RNAs:
  1. Thaw cell pellets
  2. Use Qiagen RNA prep kit.  We have lots and can get more quickly through LSC Stores.
  3. Don't use the DNase step.
  4. Elute in 40 µl H20.
  5. Measure concentration of 1 µl with Nanodrop.
  6. Run 4 µl in a 1% agarose TAE gel at 60V.
Treat to remove DNA:
  1. Use volume containing 1 µg RNA (using RNA concentration from Nanodrop)
  2. Use Ambion Turbo DNase 
  3. Use protocol in RA's notebook #1 (not missing); 2 x 30 min incubations
Check RNA quality (and concentration?):
  1. Use the 'Bioanalyzer' (high-tech equivalent of gel electrophoresis) to check the size distribution of the RNAs in each prep.  Expect to see 2 strong rRNA peaks.
Treat to remove rRNA:
  1. Use Ribo-Zero kit to remove the rRNA from each sample.
Check RNA quality (and concentration?) again:
  1. Use the 'Bioanalyzer' (high-tech equivalent of gel electrophoresis) to check the size distribution of the RNAs in each prep.  Expect to see no rRNA peaks.

Samples:
Here's the chart showing the MIV-competence samples I had planned.  I'm only going to do one set of the ∆hfq strain now, because our procedures aren't optimized for small RNAs (poor recovery and no strand information).  One of our summer-Honours students will be working on this mutant, and he can take my preliminary data and use it to help design an optimized RNAseq procedure.  So I think on Day C I'll only do the three strains (sxy-, ∆659 and ∆6759/660), and if this goes smoothly scale up to four strains on Day D.  Maybe on Day E I'll replace the ∆hfq strain with something else I want preliminary data about.


She lost me at the Central Limit Theorem

I've been saying for ages that I need to learn the statistical programing language R, so that I can work with all the bioinformatic data we're generating.  So yesterday I looked through the Coursera offerings and found an introductory statistics course that taught R (Data Analysis and Statistical Inference, taught by Mine Cetinkaya-Rundel of Duke University.  It started a few weeks ago, and I've spent yesterday watching the Week 1 videos and doing the Week 1 R lab. 

The labs are excellent.  They use a web-based R learning platform called DataCamp - each lab is a long series of guided exercises: with each exercise you're given a bit of instruction and asked to use it to display something or graph something or calculate something.  Integrated multiple-choice questions check your understanding - DataCamp automatically sends your results back to Coursera.

It's also very good that they're part of a basic statistics course, since I've always been disgracefully ignorant of this.  The video lectures are good -short but many, and aimed at the complete beginner.  I was initially quite pleased to be learning what 'marginal' means, and the differences between variance and standard deviation and standard error.  The course materials are very well designed.

But I started getting frustrated when I tried to think more deeply about quantifying variation.  I can sort-of understand why we want to know how variable the members of the population are, but this was never really explained, and I have no idea why we measure it the way we do.  To me it seems simplest to measure variation by calculating how different each individual is from the mean (the deviations), summing the absolute values of these and dividing by the number of individuals.  But that's not what's done.  Instead we square each of the deviations and sum that, to get the 'variance'.  But we don't use the variance (at least not yet), instead we take its square root, which we call the 'standard deviation' and use as our measure of variation.  Why is this cumbersome measurement better that just taking the mean of the deviations?  The instructor doesn't explain; the textbook doesn't explain.

In the first lecture of Week 3 we come to something called The Central Limit Theorem'.  This is apparently a big deal, but I don't know what the point is.  We've moved from considering the properties of a single sample from a population to considering the properties of many independent samples (size n) from the same population - I have no idea why.  The Central Limit Theorem tells us that, if we take many samples and calculate the mean of each one, the mean of these means will be the same as the population mean (is this not expected?), and that the shape of the distribution of means will be 'nearly normal', and that the standard deviation of the means will be smaller than that of the population, by a factor of 1/√n.  So what?  What do we gain by repeating our sampling many times.  Seems like a lot of work, to what end?

Then we're supposed to learn a list of conditions under which the Central Limit Theorem applies.  But without understanding the point, this was too much like rote memorization to me, and why should I bother?

Does fructose inhibit development of competence?


I gave a seminar at Michigan State yesterday, invited by the Microbiology graduate students (Thanks guys!).  While I was there I met with a research group that works on Actinobacillus succinogenes, a relative of H. influenze.  They were interested in improving the competence levels of this species, and I explained that induction of the H. influenzae competence regulon was controlled by CRP and cAMP, with cAMP levels determined by the availability of external fructose to the phosphotransferase system’ (PTS) sugar-uptake system.


The PI then asked me whether we’d tested the obvious prediction that adding frucose to the culture medium should inhibited the development of competence, and I was shocked to realize that we hadn’t.  At least I don’t think we have.  The PTS and fructose studies were done by a very competent PhD student about 15 years ago, and I’m pretty sure I would have remembered the result of this experiment, since it would either have nicely confirmed our hypothesis or disproved it.  I’ll check her thesis when I get back (I ‘m writing this in the East Lansing airport).

Assuming she didn’t do this, I’ll do it right away.  Two experiments really, a time course of competence development in rich medium and induction by transfer to MIV starvation medium.  I’ll grow wildtype cells in regular sBHI, and at OD =0 0.2 (log phase) I’ll collect them and resuspend them in either MIV or fresh sBHI containing either fructose or glucose (as control).  What sugar concentration should I use - is 0.5% standard?

Hmm, I just remembered some old experiments I did (20 years ago?) testing the effect of adding glucose to MIV  I vaguely remember that the cells became very unhappy  - did they die from unbalanced nutrients???  I know that glycerol added to MIV doesn’t have any effect on competence (very old publication confirmed by me).  I could use a different sugar (ribose? xylose?  one that we know H. influenzae can use).

Woohoo! RRResearch made a top-ten list!

If you've come from the list of The 10 Must Read #womeninscience Blogs, you might be a bit disappointed to see that the most recent posts are descriptions of day-to day work in my lab.  That's typical for RRResearch, but here are a few posts you might find more interesting:

Checking the hypercompetence mutants

Here's the results of the transformation time courses of the control strain 'K' (= KW20) and the hypercompetence mutants (RR735 and its backcross descendant RR735).

The lower graph is just culture growth (CFU/ml).  You can see that the two mutants (red and green lines) grew slightly slower than the control (blue line).  The upper graph is the transformation frequencies.  You can see that the two mutants behave identically, and that their transformation frequencies are 100-fold higher than wildtype in log-phase growth and 10-fold higher as growth slows. The dotted red circle indicates two samples that gave no transformants, so the values plotted are upper-limit estimates of the true transformation frequencies.


I also made the chromosomal DNA preps from these two mutants and from a parallel pair of mutants whose mutation has been identified (200 µl of each, at about 150 ng DNA/µl).  I set 50 µl aside for the postdoc's sequencing.