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
 * Does (v/v) mean by volume?
 * Anthony Salvagno:Good question, I don't know?
 * Brian P. Josey 14:04, 17 November 2009 (EST) According to a quick search volume to volume
 * 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.

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


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