User:Brian P. Josey/Notebook/2009/11/11

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Ant says

For the next couple of days, weeks, lives, you will be working on optimizing PCR reactions. I had been having problems with the PCR reactions I was doing and with your help I finally got one working. I want you to figure out optimal settings for the failed reactions because they have to work at some point. Of course I will guide you along the way, but this will be the first real experiments that you will be performing. Welcome to KochLab DNA!

Background

You can find any PCR reaction I have done in my notebook under the category Ant PCR (still a work in progress). Generally on each page I have the amounts of various solutions and the thermal cycler settings. This would be a good starting point. I also have some info I read up on here. Finally the internet works really well, there are some instructions that came with the Taq we use that has relevant info (see the side of the -20C freezer), and the end all be all of information is available in the lab's copy of Molecular Cloning.

Project Outline

There are 4 primers to work with:

  • F834-dig
  • F853-dig
  • R1985
  • R2008

Two are forward primers and two are reverse primers. The forward primers have the all important digoxygenin molecule. You need to figure out the best reaction conditions to get each of these to work. We have one more reverse primer coming in, but I will be working closely with you for that one. The things to consider immediately are temperature and divalent cation concentration (Mg++). Of course the other things in the reaction are important, but these have the most impact. Here is what you will do generally speaking:

  • Set up a PCR reaction. Make 5 samples (each sample is one tube with 100ul of reaction, can also do less).
  • Across each sample vary the amount of MgCl in each tube in 0.5uM increments. This will help determine how much Mg++ we will need.
  • Set up thermal cycler settings. Each time you do the reaction, you should change the temperature settings until you get something that gives a good yield. Consider the three phases of the reaction:
    • Melting - generally really high T, but just below melting so the Taq doesn't denature
    • Annealing - just below the melting temperature of the primers so they can stick to the template DNA
    • Extension - set for optimal Taq conditions usually 72-74C
  • We also want to focus on the times of each phase. The time of each phase determines how much product gets made.
  • After the reaction has reached completion you will then analyze your product on a gel, and determine amounts via the nanodrop and by mass comparison.

Hopefully by the end of this you will be proficient in PCR setup and gel analysis. Upon completion we will adapt your project further.

Recording Data

I want you to get used to making Google Spreadsheets and embedding them in your notebook for easy reading later. Your first task will be to make a basic PCR reaction spreadsheet with generic names for the DNA and include thermal cycler settings in it. From this it will be easy to make adjustments to PCR reactions down the line. You can start with one of my spreadsheets' reaction conditions and adapt.

Scheduling

With your current schedule, it will not be possible to do both the PCR and gel in a single day. I assume on most days you will only be able to get the reaction started. In this case I will put your samples in the -20 when the reaction is done, and then on the next available day you can do the analysis.

Getting Set Up

When we begin I will show you where everything is kept (you have seen it once before) and how you should maintain it. We will give you your own freezer box for your samples and your samples only. No one else is to go in there, not even me unless I ask you. We will also set you up with your own tips, tubes, and chemicals if need be. We will clean up your space to make it adequate for doing molecular biology!

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