User:Brian P. Josey/Notebook/2009/12/03

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Everything That Needs to Be Done

Because I have a lot to do over the next couple of days, I just want to have a list to cover everything that I want to do and my approach to it. I want to practice the tethering procedure at least one more time before Tuesday. This doesn't really take too much time, and I imagine that I can do one during the morning on either Friday or Monday, so I will hold on to it for today. I also really need to run a PCR of the double stranded DNA, I don't need too much time to set the reaction up, but the reaction itself can take a while. I would like to get that one taken care of today before the lab meeting. I also need to go through my notebooks entries from the week of Thanksgiving forward to clean them up and put all the data in. Unfortunately, with my schedule I have a hard time getting the pictures of gels into my notebook before I have to run to class. Anthony has them all in his notebook for the corresponding page, but I would like to copy them over to my own notebook with my own comments. So the plan of attack for the next couple of days is:

  • Set up procedure for double stranded DNA PCR
  • Run double stranded DNA PCR
  • Run a gel to check the PCR
  • Write up my own notes for the tethering procedure
  • Practice tethering process
  • Film tethers and get hands on with the microscope
  • Clean up notebook
  • Go through papers

Double Stranded DNA PCR

This PCR was modeled off of one that Anthony did with Diego over the summer. The original experiment is here. There were twelve samples, labeled 1A-6A and 1B-6B. The difference between each of the samples is the bio and dig primers, and the concentration of Mg2+ ions. There are two different dig primers, three different bio primers and two different concentrations of Mg2+ ions. A tube was made for every possible combination of these as illustrated in the table. The samples were made by first mixing two master solutions, one for each concentration of Mg2+ ions that did not contain either of the primers. The primers were dispensed into their proper tubes followed by their respective master mix solutions. The exact reagents and what tube they were placed in are:

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The times for the Thermo Cycler were:

Stage Temp (C) Time (mm:ss) Reps
1 94 4:00 1
2 94 1:00
60 1:00
72 5:00 35
3 72 9:00 1