Wednesday, July 15, 2015

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

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