RAPD-PCR Update (July 24)

As our research into the CBAY samples moves forward, we’ve continued our use of RAPD-PCR to look at banding patterns.

After our last reported run, we know that CBAY1 amplifies with the OPA-9, OPA-13, and CRA-22 primers. The dilution that worked best was 10^-2. In order to verify our results, we’ll run duplicate reactions using the 3 primers at the 10^-2 dilution, once again using HotStart Taq.

We set up four replicate reactions for each primer. For OPA-9 and OPA-13, we’ll pool the four reactions into two, hopefully increasing the strength of our bands on the gel. For CRA-22, we’ll pool two of the reactions into one, leaving us with one 2x reaction and two 1x reactions.

The lane order was as follows (lanes 1-10 are the first row of the gel; lanes 11-20 are the second row of the gel):

1. ladder
2. OPA-9 (2x)
3. OPA-9 (2x)
4. OPA-9 (+)
5. OPA-9 (-)
6. OPA-13 (2x)
7. OPA-13 (2x)
8. OPA-9 (+)
9. OPA-9 (-)
10. ladder
11. ladder
12. CRA-22 (2x)
13. CRA-22 (1x)
14. CRA-22 (1x)
15. CRA-22 (+)
16. CRA-22 (-)
17. blank
18. blank
19. blank
20. blank

This gel looks very good – we have strong bands in one of the OPA-13 lanes and in the 2x lane of the CRA-22 lanes. All positive controls produced bands, and the negative controls did not result in bands.

We’ll continue discussing and analyzing these results. In the meantime, we’ll move forward with running the CBAY2 samples under the same setup as the CBAY1 samples.

 

Lab Notes – July 22

Today, our beta-Agarase came in, giving us the ability to degrade agarose in old plugs, as well as the screwed up VC’s I tried to create for PFGE last month.

First, we’ll purify out the agarose from these plugs, then purify the DNA. We’ll then have more DNA to work with in RAPD. Always a good thing, especially since our Chesapeake VC’s are running low.

Also, we’re currently looking into the idea of reusing old anodiscs. We have a large collection of slides that have been made over the past school year/summer. By now, they’re unusable for microscopy as a result of fading; also, we’ve already taken pictures of these filters, making the retention of hard copies no longer necessary.

We’re not sure how to proceed with the cleaning procedure, but we’ll most likely use a combination of ethanol and boiling water to remove the stain and particles from the filters. It’s a low-risk, high-reward experiment. If successful, we’ll no longer need to wait for the backorderd filters – and we can continue with our Lake Matoaka Study, as well as my Outer Banks Study.

 

RAPD-PCR: CBAY1, July 22

Yesterday, labmate Dan Kiernan and I began a run of RAPD-PCR (Randomly Amplified Polymorphic DNA Polymerase Chain Reaction) on the Venter Institute’s Chesapeake Bay Samples (CBAY1, CBAY2).

In short, RAPD-PCR amplifies DNA in a random fashion, using primers that don’t necessarily have a match on your sample sequence. We used four primers for this experiment: OPA-9, OPA-13, CRA-22, and CRA-23. For our reactions, we also used Hot Start Taq; Taq is the enzyme that adds the basepairs to the DNA strands. Hot Start has a protein associated with it that will dissociate once a certain temperature is reached (in the thermocycler), helping to prevent primer dimers and other mis-priming events.

We used dilutions of 10⁰ (undiluted template), 10^-1, and 10^-2, as well as a positive and negative controls.

We’re hoping to see varied banding patterns, giving us an idea of what’s in the samples we received from San Diego.

The gel was loaded in the following sequence (lanes 1-12 represent the first row; lanes 13-24 represent the second row):

1. ladder
2. OPA-9 :0 Dilution
3. OPA-9 :-1 Dilution
4. OPA-9 :-2 Dilution
5. OPA-9 :+ control
6. OPA-9 :- control
7. OPA-13 :0 Dilution
8. OPA-13 :-1 Dilution
9. OPA-13 :-2 Dilution
10. OPA-13 :+ control
11. OPA-13 :- control
12. ladder
13. ladder
14. CRA-22 :0 Dilution
15. CRA-22 :-1 Dilution
16. CRA-22 :-2 Dilution
17. CRA-22 :+ control
18. CRA-22 :- control
19. CRA-23 :0 Dilution
20. CRA-23 :-1 Dilution
21. CRA-23 :-2 Dilution
22. CRA-23 :+ control
23. CRA-23 :- control
24. ladder

The gel looks good, and we had priming sites for 3 of our primers (OPA-9, OPA-13, CRA-22). Positive control bands appeared for all 4 primers. The only red flag is bands appearing in the negative control lanes for OPA-13 and CRA-22. This could be a result of my poor pipetting abilities, so we’ll take the gel for what it is: an overall success that will lead to replicate reactions.

That’s what we’re working on now: replicating our work. Since the bands only appeared in the -2 dilution lanes, we’ll use -2 dilution, OPA-9, OPA-13, and CRA-22 to repeat our work. Hopefully, our bands reappear in each lane on our next gel.

I’ll keep you posted.

 

What’s To Come

Looking forward to the remaining 3 weeks of summer session research, there’s still much to be done. We’re making progress on the Matoaka project, and we’re starting microcosm work with the CrimD phage (isolated here at William & Mary).

Unfortunately, however, the lab is still without Anodiscs or a comparable slide-making filter. We’re stuck on the Matoaka project until we find a replacement or the back-ordered discs come in – without them, we can’t enumerate our viral samples, making the goal of tracking community changes impossible to reach.

The same goes for my Outer Banks study – I can’t move on unless I have filters to look at my samples. So until the Fall, that project will remain incomplete.

For PFGE, I believe to have optimized the conditions for our needs and samples. There’s little left to tweak at this point.

So, my concentration will lie in DNA recovery from agarose plugs. As mentioned very briefly in an earlier post, the Venter Institute in San Diego sent us VC’s from their study of the Chesapeake Bay. In an attempt to maximize resolution with PFGE, Dr. Williamson and I loaded a smaller volume of higher concentration agarose in the plugs – allowing us to load more of the VC. The attempt ended in failure, as the agarose solidified too quickly for us to work with.

We’ll recover the DNA from the plugs, giving us more DNA to work with for RAPD-PCR and other DNA-based techniques. Hopefully, we can give the Venter Institute some meaningful results.

For now, we’ll move forward where we can with what we can. Fall will be an interesting time for research in the Williamson Lab.

 

Summer Profiling PFGE v.2 Update (July 15)

Well the new gel turned out quite well. The gel lanes are the same as the previous run (see this post for details).

The new conditions (run-time: 19.5 hours; initial switch-time: 0.3sec; final switch-time: 6.5sec) have achieved the desired effect: the bands at the upper and lower sizes have resolved a bit more clearly, and the entire gel has better separation.

We can also see some slight variation in the banding patterns from month to month and from site to site. This suggests that the viral community is indeed changing over time and space, which is what we’d like to prove with our work this summer.

Until we sample in August, PFGE is finished for right now. Hopefully these conditions are the final optimized ones.

 

Summer Profiling PFGE v.2 (July 15)

After discussing the issues with Dr. Williamson and conferring with Birren and Lai’s “Pulsed Field Gel Electrophoresis: A Practical Guide”, we decided to re-run the samples from the previous gel.

This time, we’ve altered the run conditions yet again: a run-time of 19.5 hours, an initial switch-time of 0.3 seconds, and a final switch-time of 6.5 seconds. We hope this will help resolve bands at the lower and higher ends of the spectrum, as well as separate the individual bands a bit.

The gel is almost done running; when completed, I’ll stain and visualize it. Stay tuned!

 

Summer Profiling PFGE (July 14)

Following up on my previous post, I completed the rinsing of PFGE plugs, prepared the 1x PFGE-Certified Agarose Gel, loaded the plugs into the wells, then loaded the kb ladder and lamdba genomes.

We ran the gel under the same conditions as before – 18 hours at 14°C with an initial switch-time of 0.5 sec and a final switch time of 3.0 sec.

The order of the samples on the gel is as follows:

1. 1 kb ladder
2. digested lambda genome
3. lambda genome
4. March Keck Pier VC
5. April Keck Pier VC
6. April Matoaka Spillway VC
7. May Keck Pier VC
8. May Matoaka Spillway VC
9. May Matoaka Inlet VC
10. June Keck Pier VC
11. June Matoaka Spillway VC
12. June Matoaka Inlet VC
13. July Keck Pier VC
14. July Matoaka Spillway VC
15. July Matoaka Inlet VC

Worth noting is the fact that the digested lambda genome is nowhere to be found. It should be noticable, as it marks the kb lengths in between the ladder and the genome.

Also, July is seemingly empty. This is also strange, but not inexplicable.

For the July samples, we’re using new filters to capture the viruses, and they work differently than our old Vivaflow filter. This could have something to do with the lack of virus particles; my labmate and water guru, Dana Hardbower, even expressed her concern over the new filters to me. That’s a possibility. On top of that, our lack of usable filters makes it difficult to guarantee that we even have particles. I may prepare a wet-mount slide later just to be sure (note: wet-mount slide is placing samples directly on a slide, then viewing under the scope).

It is positive to note that there does seem to be changes in the community from month to month, site to site. This is what we’ve hoped to see, as it proves that the viral community is a dynamic, changing entity. Excellent news.

For now, I’ll discuss the issues with Dr. Williamson, and hopefully we can devise a solution. I’ll keep the blog updated with developments.

 

To-Date PFGE Run

Sorry for the short hiatus – family time at the beach took priority over science, if only for the week.

I returned to lab yesterday, and my current PFGE-related project is to run every viral concentrate from Lake Matoaka so far and compare the banding patterns. These include the Keck Pier samples for March, April, May, June, and July; the Matoaka Spillway samples for April, May, June, and July; and the Matoaka Inlet samples for May, June, and July.

Since we’ve already cast the June VC’s into InCert Agarose plugs, we only have 9 to do on this run.

Right now, I’m sitting in lab waiting for the plugs to finish rinsing in TE Buffer. Unfortunately, I am out of TE Buffer and still have one more rinse cycle to go. Due to poor note-taking, I do not have a recipe for TE – in fact, I don’t even know what the concentration is, or even the molarities and pH’s of the ingredients.

So, I’ll store the plugs in the TE they’re being rinsed with at 4°C until Monday. Sometimes science just doesn’t go the right way.

 

Outer Banks Note (July 4)

After being back to the beach today, I must report an error in sampling.

The location I used for ‘High Tide’ is actually the high water line for storm swells. ‘High Tide’ is actually what I sampled as ‘Medium’.

Luckily for me, I took two sub-samples, so there still is a ‘Medium’ location. Nothing major, but important and worth noting. I’ll update the map shortly.

 

Lab Update (July 03)

We’ve hit a bit of a roadblock in lab.

A lot of the research we carry out here revolves around our ability to make slides using ANODISCS for use in microscopy. These little filters capture our viruses and bacteria, allowing them to be stained and mounted on a slide.

Recently, however, we’ve found that many of our slides have contamination on them. After testing multiple variables, including the sterile water we use for slide making, the SYBR Gold, and the vacuum manifold itself, we’ve come to the conclusion that our Anodiscs are contaminated. This is very unfortunate, since the discs are on back order (since March), and we have no suitable alternative right now.

We’re waiting on an order of comparable filters, but until then, the Lake Matoaka experiment is iced (for the most part), as is my Outer Banks project.

So for now, we’re in planning stages for the following week. I will be away at the beach until July 9, so until then , enjoy the summertime!