User:Brian P. Josey/Notebook/2009/11/13

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PCR and Some Ideas

I managed to read some more from Molecular Cloning last night about the PCR. Despite being such a large book that is dense with material, it is surprisingly easy to read and interesting. I will update some notes a little later on today or over the weekend. I also want to gather my ideas together onto a couple of pages in the notebook.


Buffers

Taq:

  • 20 mM Tris-HCl (pH 8.0)
  • 0.1 mM EDTA
  • 1 mM DTT
  • 50% (v/v) glycerol
  • Stabilizers
    • Sounds mysterious, and I am curious to know what they are

10X PCR Buffer:

  • 200 mM Tris-HCl (pH 8.4)
  • 500 mM KCl

Practice PCR

Just for the sake of practice, I made a generic PCR spreadsheet. I designed it to include concentrations of Mg+2 to go from 1.0 mM to 5.0 mM.

{{#widget:Google Spreadsheet

key=tnDEmLGIKdfnJ9oKx41lY2A width=500 height=300

}}

Ant says

Anthony Salvagno 16:07, 13 November 2009 (EST): Looks great! Some quick notes...

  • You want to state template DNA in terms of ng/ul which in our case starts at 1.5ng/ul so your desired concentration should be adjusted accordingly
  • You state your desired buffer concentration is 10mM, but 10mM what? I think you should just record starting at 10x and want 1x, unless you want to include the components of the buffer.
  • I think you made a little mistake in your calculation of MgCl2. You say you want 1-5mM from 50mM, but you put 1ul in 100ul which is .5mM. The minimum you would want based on your desired conc. is 2ul.

With all that said I think you are off to a great start. I think you should set up a PCR plan maybe at some point this weekend and do it on Monday. Pick 2 primers (say the F834-dig and the R1985) and do different MgCl2 concentrations ranging from 1-5mM in different tubes.

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