Lab Post #3 (8/31/16)

Today we prepared the PCR to modify the SAK1 gene. All of the reagents had to be kept in ice to make sure that they wouldn’t denature. Here is the amount of each reagent that we used:

  • 5 microliters of the template DNA for SAK1
  • 40 microliters of nuclease-free water
  • 2.5 microliters of our Forward Primer
  • 2.5 microliters of our Reverse Primer
  • 2.2 microliters of the DNA Polymerase
  • 44 microliters of a buffer
  • 6.6 microliters of DMSO (which dissolves both polar and nonpolar compounds)
  • 4.4 microliters of dNTP (which is a nucleotide)

The reason that some of these numbers do not match the ones on the PCR Protocol that is attached at the end of this post is because the Polymerase, DMSO, dNTP and buffer were part of a master batch that each group doing their experiments used. Those proportions were multiplied by 4.4 so that each group would receive 13 microliters of this solution (with some extra left over in case of an error). Afterwards, each group added their specific primers, DNA template, and water.

Because the template DNA for SAK1 was at a concentration of 202.2 nanograms/microliters, a dilution was needed to reach the appropriate concentration of 20-250 ng in solution. We combined 5 microliters of nuclease-free water with 5 microliters of SAK1 template DNA. We then took 2 microliters of this dilution and pipeted it into the tube with the 13microliters from the master batch. We then added 35 microliters of water so, in total, the solution was 50 microliters.

This solution was then put into the thermocycler.

That’s it for now. Until next time, check out the photos below & the attached file for the entire procedure.

image
This is the ice bucket where all the reactants we used were kept in order to prevent denaturation.
image
Here’s the mixing of the reagents for the “master batch” to split with groups 1, 2, and 3. This does not include our primers, water, or DNA template.
image
Here is a close-up of the mixing of all the reagents, with the exception of the water, primers, and the DNA.
image
Here is the centrifuge that mixed up our water and SAK1 DNA Template for our dilution.
image
This is the thermocycler, which heats up whatever is inside very quickly and then cools it down for a bit and repeats this for a number of cycles in order to create double strand breaks and cut out our selected gene.

PCR Protocol

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