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Addition of pcr reagents to master mix - What order is best? (Dec/19/2005 )

Hi All,

What is the best order in which to add reagents to make the PCR master mix? I have been adding them in this way:

  • 1. Buffer
  • 2. MgCl2
  • 3. dNTP's
  • 4. AmpErase UNG
  • 5. DNA Poly
Then, I add nuclease-free water to my primer mix tubes, add the Master Mix from above, and then add my primers and probes and finally template.

I just read, however, that the MM should be dispersed into tubes, followed by MgCl2 and template.

Other than making sure not to add enzyme to a mix without buffer, does the order make a difference? Sorry if this question is very naive!!



I'd do:

1. ddH20
2. Buffer
3. MgCl
4. AmpErase UNG
5. dNTP's
6. Primers
7. Polymerase

aliquot out resulting MM.

Really shouldn't matter that much, though...