We are getting close to sequencing!

A few of the PCRs last week didn’t work so I’m finishing up the last PCR B’s right now that we’ve had to redo and then all we have to do is Gel confirmations. If I counted correctly there are 24 confirmations to do. I would like to do one now, but I have to catch the bus in 30 minutes and I wouldn’t finish in time 😦   I plan to come in on Friday and possibly Thursday as well and hopefully as a group we can completely finished and get our samples in the sequencer by the end of the week!

 

Then comes the hard part! Data analysis. I learned a lot about this portion of the project while I was preparing my part of the lab meeting presentation a few weeks ago so hopefully that knowledge helps me understand what’s to come. Like most things, I’m sure it’s much easier said then done!

PCR B

Today I did 32 PCR B’s. It may sound like a lot but PCR B’s are extremely easy so it went by pretty quick. I also messed up on updating the google doc and had to throw away three PCR A’s that I did last week. Not a huge deal, but kind of annoying!

 

Right now we have a lot of Gel Confirmations to do. I counted and there’s 48. So to whoever is in the lab next… have fun! 😉  I’m hoping they all come out good so we can get some sequencing done!

Today in the lab

Today I primarily focused on reorganizing some of our stuff. First, I went through our two boxes of extracted DNA and put them in numerical order by sample ID. I think at one point they were in order but with all the PCR redos they’ve gotten quite mixed up and I’ve found it hard and annoying to find the right samples. The first box has any samples under #160 and the second box is any number above #160. The samples are also in order in each box, but I have a feeling that won’t last long 😛

 

Next I reorganized our PCR box. This didn’t take long. All I did was check to make sure all the forward and reverse primers had stuff in them, replaced a couple empty ones, and put them in order in the box. I did have one concerning discovery though: One of the primers said F2 on the side but F3 on top. I threw that tube away to avoid further confusion.

 

Lastly, I went through each box of samples from the minus 80 degree freezer and wrote the sample ID range of the samples in that box. For example I would have written “#’s 200-300” for a box with all samples between 200 and 300. It was a bit difficult to write on the boxes because they were so cold!

PCR A, PCR A, and more PCR A…

I’ve spent the week doing PCR A! So far I’m up to 40+ PCR A reactions. I finished up the 20 remaining samples needing PCR A this morning. After losing track of where I was in the PCR A process on Tuesday (and having to restart half of them) I developed an extremely organized way to do today’s set of samples! I meant to take a picture of the chart I made and used and post it here, but I forgot 😦

 

We are almost done with our preliminary set of samples to be sequenced! Assuming all the PCR A’s work, we will have quite a bit of PCR B to do (which is a lot quicker than PCR A!) and then gel confirmation, quantification, and dilutions to 1 ul. Maybe we can get it done in the next week.. too optimistic? We’ll see!

 

On a side note, David and a few others get to go to the Kings vs. Spurs game in San Antonio to collect samples for their space project. What a cool opportunity! (Say hi to Tim Duncan for me!)

Organization plan of attack!

One thing I would like to do tomorrow is go through our boxes and organize all the DNA, PCR reactions, and final products by sample ID #. I also want to write on the boxes of DNA and PCR reactions which numbers are in that box. For example, we have two DNA boxes (and probably more to come), and it can be quite tedious finding a DNA sample when you don’t know which box it’s in and the ID #’s are written very small on the lids of the 2mL tubes. (You would feel the same way if you had my terrible vision!)  I also want to take the original samples out of the freezer and write on the box which samples are in that particular box. Those samples are in the negative 80 degree fridge and it can be quite painful sitting there opening and closing box after box looking for the write samples. Hopefully I get a chance to do that and hopefully it helps 🙂

Lab presentation last Friday

Unfortunately I missed our lab meeting presentation last Friday, due to a doctor appointment taking longer than expected, but I did learn a huge amount of what’s to come in the data analysis portion of our project (basically everything that happens after sequencing). It’s going to be a lot of work, from assigning OTUs to building phylogenetic trees and much more!

 

For now we will be continuing our normal lab work. We have made a few modifications to speed up the process. For example, for PCR A, we are adding a more rough amount of DNA rather than calculating it all out, which takes a good amount of time. Note: this does not make our end results less accurate.

 

I’m excited to finish our first set of time intervals for Coral Pond 1 so we can get them sequenced and start analyzing!

Pipet or Pipette?! And some updates on our project :)

First of all, how do you spell the thing?? I’ve seen it both ways.. pipet and pipette. I’m thinking it’s pipette because it doesn’t have the red squiggly spelling error sign under it. Just wondering!

 

Now for the good stuff! We’ve been rocking and rolling in the lab. (Except for the week I came down with the flu) We have so many samples and it would be a bummer to do EVERYTHING (DNA extractions, PCR, Gel confirmation, etc) for every single sample and have disappointing results. While we are optimistic about our results, we are currently preparing samples from the first coral ponds at different time points for sequencing just so we can see how our results look in terms of succession of the microbial community in the pond. If we get cool results (crossing our fingers) we’ll go back to the pipettes and prepare the rest of the samples!

 

We’ve got a lot of work to do but we are making progress. We are presenting to the lab next Friday about our project. I am really excited to get feedback from experts who know a lot more than me!

Back in business

We’ve been sampling every day for Coral Ponds 1 and 2. We are up to almost 500 samples… which means we have a lot of DNA extractions to do.

 

I’ve noticed a few things in the last month. The coral used to be white, but now it is covered with a reddish algae. This is good because clearly things are changing, which is what this study is about! If macroscopic things (for example, the visible algae) are changing, then the microscopic things (microbial community) are most definitely changing as well!

 

On a side note, Matt and I discovered the time it took for a full round of sampling at maximum efficiency. With water filtering being the rate limiting step of our sampling process, we focused on wasting no time with the filtering. By this I mean, we had the next liter of water ready to go instantly after the current liter was done filtering. We also set a timer to remind us to keep adding water to the filter. Out of curiosity, I timed our last filtration and it took 17 minutes and 30 seconds. Overall we were able to complete the sampling in about 1 hour and 50 minutes. Being the mathematician that I am (just kidding), I assumed each filtration (6 total) took about 17 minutes and 30 seconds and calculated the time that water was not being filtered from the time we started to the time we finished: 5 minutes! I’d we were pretty productive.

Sampling yesterday

Yesterday we did PM samples on both Coral Ponds after they were inoculated. Our rate limited step (the water filtrations) took even longer because there was so much sand in the water. The sand would collect on the filter, causing the water to pass through much slower. We didn’t filter the water from Coral Pond #2 because there was so much sand that I think the water filtrations would have been unsuccessful. We did take water samples and left them overnight so that the sand would settle and David filtered the water this morning. We did run all water chemistry tests on both Coral Ponds last night.

Because our nitrite meter only measures up to 200 ppm, I did a 1:4 dilution for the water from Coral Pond #1 and a 1:10 dilution for the water from Coral Pond #2 in order to get readings within the range. Then I multiplied to get the correct value.