Purifing and a little movie magic

Objective: 

Take the product from July 22nd purify it using ULTRAFREE DA DNA extraction from agarose ref#42600. After purification test samples in the nanodrop to check quantities of DNA. 

Start reviewing the videos and collecting data from one of the sea pens. watch every 7th video recording behavior hourly on excel spreadsheet created on July 13th. behavior noted: if the animal was extended, unexteneded, if a peristaltic like wave motion was visible, if the animal was visible and of the animal had uprooted itself. The videos cover from 08/14/2014 to 01/24/2015. I used quicktime to view the videos since it allowed me to scroll in easily through the videos. 

Results: 

Sequencing

The purification was successful but the nanodrop recorded low amount of DNA (show below). I am not sure if the samples will be able to be sent out for sequencing but I will talk to Sam to verify

Behavioral

I was able to collect data for 21 days focusing on one of the sea pens. the first to days (time-lapse 1 and 7a) I focused on the sea pen that was positioned in the front right bucket, in video 7b the arrangement was changed and I followed to animal in the front center. I was watched video 1,7,14,21,28,35,42,49,56,63,70,77,85,92,99,106,113,120,127 though after 99 the videos became very dark making it very difficult to make out animals often only able to get 1 data point. Later on in the experiment I did reduce the lighting which seemed to increase animal activity though made it to difficult to obtain video data. 14 of the videos did give usable data. Viewing the videos took about 2 hours though the final videos were quicker since I could not see much. If I continue viewing them on a regular basis I should get faster and may be able to increase the animals that I am recording but I will speak with Dr. Roberts on Friday to verify how he wants me to proceed.

Sanguine PCR of a PCR

Objective: Taking the product of the PCR run on 15, july 2015 and running it out on a Gel to see if the PCR of the PCR product from 1, july 2015 was successful. Hopefully it was amplified enough to be able to sequence. Sam is gone for the week so I will be able to get verify next week.

Gel:
50ml 1x TBE
.5 grams agarose (1%)
5 ul athidium bromideGel run with ogene ruler 100 bp dna ladder (thermo scintific #sm1143 lot # 00189314)
(PCR product 1 and 2 were taken from the combining of two gel bands from previous PCR)

1 5ul Ladder
2 PCR product 1
3 PCR Product 2
4 H2O control 1
5 H2O control 2

I was also having trouble interpreting the results from the Muscle Alignment and so I talked with Jake who recommended some names (DR. Carrie Naish, Charlie Waters, and the Seebs) to try to get in contact with that have more experience with Geneious.

Seeb, Jim	jseeb@uw.edu		175B MAR
Seeb, Lisa	lseeb@u.washington.edu		175A MAR

Naish, Kerry

knaish@u.washington.edu

209 MAR

Results:

The PCR was successful the bands in row 1 and 2 that had the combined PCR product from the previous PCR were amplified correctly and show up much brighter than the previous. I removed the bright bands from the 1000 bp mark on the ladder and put them in separate tubes. I noticed a mistake that I had been using another ladder diagram that shows the bands at 1000bp which was incorrect the correct is shown on this I will replace the images on the others with the correct ladder. i still show that the sequencing that we have been getting is still half of what is being sent not likely reason why the length is so much smaller. 500 bp is still smaller than the expected ~850 that I am looking for. Supports the idea that new primers may be needed. All in all a nice looking PCR I believe but will verify later and hopefully get this run off for sequencing next week. Jake brought up that the bands were extremely close together so without good separation that it would be difficult to know that the bands that I cut out are the strands that I am looking for. When the bands are so close together it is very easy to get "junk" other than what I want. This may mean that sequencing may not be an option.  also noticed that when taking a picture with my phone it was easier to take the orange filter and lay it on the cover of the machine. My phone seemed to focus a lot quicker and took a better picture then just holding the filter directly on the camera phone.

Objective: Wanted to go over my notebook with Sam and identify any issues, as well as look at the excel spreadsheet that I created for taking data from the videos. I been talking with Dr. Alaron Lewis from the University of Washington Bothell campus about possible issues that she could see occurring with my research since we have had issue getting a clean sequence with the Sea Pens. she brought up a lot of issues that I wanted to go over with Sam.

E-mail Discussed: 

Hi Jon.

    So, are you purifying your PCR every time before you send it for sequencing? 

Have you ever run out some of the purified stuff to figure out how much you got out? 

Have you ever sent in the PCR without purifying? 

Are you using the same primer for sequencing that you used to PCR? 

I think that the issue here is that your sequencing reaction is not working, and my best guess is that your purification of the PCR is decreasing your yield so much that you don't have enough template to sequence.

You can clean the PCR without gel purifying it so that you get more yield.

Also, are you running the sequencing reaction or are they 

In all of the sequences I looked at you have some weird peaks at 80 bp, which often indicates an excess of terminators in your reaction (see below) 

Why do massive peaks occur ~50-70 bp into my data?

If excessive amounts of unincorporated dye terminators remain in your samples after they have been cleaned, they will appear as massive peaks ~50-70 bp into your sequence data. When the levels are especially high, a secondary peak will occur ~50 bp farther downstream of the primary peak. 

So, I think you need to go back to the PCR and run some gels on your cleaned stuff before you send it for sequencing. 

You might also think if you could use a primer that was different from your original PCR primers.... 

One possibility is that you do a cloning reaction to put your PCR reaction into a plasmid, and then you can isolate that plasmid and sequence it. This would allow you to get a lot more DNA.  

Your PCR looks like it worked, but I think your sequencing reaction is NOT working. (That is why your Data makes no sense when you blast it...the bases that you are getting are essentially artifacts). I think you need to go back to the PCR and really make sure that you have a lot of very clean DNA to put in your sequencing tube. 

Dr. Lewis had recommended that I rerun the PCR product produced on july 1st 2015 to try to increase the product available for sequencing.

Results: Learned that I need to incorporate more information into my note book. I realized I was missing some key information that may make things difficult for me to remember in the future. Sam said that the data sheet I created looked good but I should try working with fake data to make sure that when I got to graph everything will work out. I decided to randomize that dates that I choose when watching the videos since daily data on the statues of the animals was not taken. Took the DNA that had been produced and started a 50 ml PCR 

PCR measurements. 

DNA  10 ul

Apex red 25 ul x 4.4 reactions (.4 added for error) = 110.00 ul

Forward Renilla Primer 1 x 4.4 = 4.4 ul

Reverse Renilla Primer 1 x 4.4 = 4.4 ul

H20 13 ul x 4.4 = 57.2 ul

individual total volume 50ml to each vial

Behavioral and Seqencing work

Objective: create a excel spreadsheet to use for the behavioral section of the experiment. Decided to choose one animal throughout the experiment and choose 1 or 2 days a week at random. The seapen located in the front center was almost always visible throughout the experiment so would be a good base for the initial collection of behavioral data.

Sequencing organize that data so it is more accessible in the notebook.

Results :

Location of the Sea pen videos:
http://eagle.fish.washington.edu/whale/index.php?dir=evocam%2Farchive%2Fseapen%2F
all videos are made up stills taken every 4 min.

Uploaded first draft of behavioral data sheet to eagle
http://eagle.fish.washington.edu/scaphapoda/index.php?dir=Jonathan%2F











Decided to take data from each hour point and note  if the animal was extended, unexteneded (buried), if it showed the wave like motion in its upper rachis that was observed when reviewing videos. and if the animal was visible or uprooted.

Muscle alignment : decided to run another type of alignment that I had used for previous work and noticed that my sequences lined up better with the initial sequence used to create the primers (shown in yellow). Red lines represent areas of the sequence that were trimmed. Consensus strand shows solid similarity up to ~200 bp then changes into sporadic similarity from ~250bp to ~400 and ~500-~600 bp. A28028 was the Renilla reniformis sequence used to create the primers shown with a yellow band bellow it (luciferase CDS)

Geneious sequencing: alignment done on June 29th that shows very little overlap. The Renilla reniformis sequence had no overlap with any of the sequences shown from ~200 to ~1200 bp all other sequences follow.

individual Sequencing

Detailed information for each of the individual sequences used in the alignment. Red indicates trimmed section and shades of blue show strength of accuracy for each base pair. Darker blue shows a higher percentage of accuracy. The name of the sequence used is to the left which can be referenced in the table above

Alignment of sequences: raw data of 11 sequences alligned excluding penF-010Luciferase_F which did not overlap with any of the other sequences shown below. 

Without penF-010Luciferase_F

with penF-010Luciferase_F

Gel run

Objective: Run the PCR product from 6/30 out on a gel to verify that the last PCR was valid since the results were not what we had expected to find. After running out on a gel Purify using ULTRAFREE DA DNA extraction from agarose ref#42600 LOT# R5DA8835.

Results: The bands were not extremely bright but still had product that was consistent with previous PCRs. Removed the bands and purified them. Will have sam send them out for sequencing.