PCR Master Mix Recipes
Master Mix Cocktail (first round PCR)
|Reagent||Volume||Extra Volume 1||Extra Volume 2||Ex: 8 reactions + 2|
|dNTPs||9.0 µl||90 µl|
|MgCl 2||4.0 µl||5.0 µl||7.0 µl||40 µl|
|PCR Taq 10X buffer||3.0 µl||30 µl|
|ITS1 forward primer||2.5 µl||25 µl|
|0599 reverse primer||2.5 µl||25 µl|
|Taq polymerase||0.2 µl||2.0 µl|
|Nuclease-free water||24.8 µl||23.8 µl||21.8 µl||248 µl|
|DNA template||4.0 µl||4.0 µl (each tube)|
Each reaction (one spore sample) will have a final volume of 50 µl. The reagents in this recipe are defined for ONE REACTION. The final master mix volume will consist of all reagents in one reaction multiplied by the number of samples to be amplified PLUS TWO EXTRA VOLUMES (to allow for pipette error). There are two exceptions: (1) the DNA template is 4 µl for the total master mix volume and (2) nuclease free water is whatever volume brings the total in ONE REACTION to 50 µl (see modifications in recipe below). Deviations from the reaction mixture specified below usually involves changes to magnesium chloride concentrations. In our experience, deeply rooted clades ( Ambispora, Paraglomus, Claroideoglomus) amplify just fine with 4 µl (1.5 mM), but Gigaspora species require 5 µl and Rhizophagus, Funneliformis, and Septoglomus require almost twice as much (7 µl). NEVER vortex the Taq polymerase or the master mix after the Taq polymerase has been added.
All of the reagents EXCEPT the DNA are pipetted into a 1.5 mL tube and then aliquoted (46 µl) into eight 0.2 mL tubes (using the example in the table above). Once this is done, then 4.0 µl of DNA from a spore extract is added to one tube.
By watching the tube contents carefully, the DNA can be seen as it is pipetted into the master mix already in the tube. Careful observation ensures that the template is in the master mix and not stuck on the wall of the tube or on the transfer pipette.
Master Mix Cocktail (second round PCR)
|Reagent||Volume||Extra Volume 1||Extra Volume 2|
|MgCl 2||4.0 µl||5.0 µl||7.0 µl|
|PCR Taq 10X buffer||7.0 µl|
|0061 forward primer||2.5 µl|
|FLR2 reverse primer||2.5 µl|
|Taq polymerase||0.2 µl|
|PCR product||1.0 µl|
|Nuclease-free water||23.8 µl||22.8 µl||20.8 µl|
In this round, the DNA template is the product from the first round of PCR amplification (less needed). The same adjustments to magnesium chloride are made, depending on genus, as those made in the first round above.
The same procedure described above is followed again. All reagents EXCEPT the DNA are pipetted into a 1.5 mL tube and then aliquoted (49 µl) into eight 0.2 mL tubes (using the same example in the table above). Once this is done, then 1.0 µl of DNA from the first PCR round of a spore extract is added to one tube.
Watching the tube contents carefully as the DNA is pipetted into the master mix is an important verification step here because the volume of DNA is so small.