TITLE: Preparing 1x PCR Master Mix
AUTHORS: Jenny Molloy, Stephane Fadanka, Nadine              Mowoh, Cordellia Cordellia Fulai

[DESCRIPTION]
This protocol documents the production of BenBio 1X PCR Master Mix "Wet" and "Dry" formulations including the different colors of the Wet formulations (Rubis or oink and Saphir or blue). This method uses  OpenVent cellular reagents. The PCR master mixes are then stored for subsequent PCR reactions.

Cellular reagents are defined as common molecular biology enzymes expressed in E.coli but not subsequently purified before use i.e. dried E.coli cells are used as the reagent (Bhadra et al (2018)). 




[BEFORE_START]
Ensure that the cellular reagent (OpenVent enzyme) to be used is available and has been pre-tested for functionality.
Ensure that all the other components needed to prepare the PCR mix are available and free from contaminants..

[GUIDELINES]
This protocol describes the steps in preparing and testing the functionality of 1X PCR master mix.

[STEPS]
SECTION: Cellular reagents preparation
1. 
 

 Prepare a fresh batch of cellular reagents (OpenVent) following BenBio protocol for plate protein expression on autoinduction media.

SECTION: Functionality test
2. 
Remove the enzymes from storage, reconstitute and test for functionality as described in the BenBio protocol using the specific test that apply for cellular reagents (OpenVent enzyme).

SECTION: Preparation of 1x PCR Master mix formulations
3. Preparing the work surface and materials:
 Clean the working surface first with 1:10 dilution of Bleach, then70 % (v/v) Ethanol
Clean the micropipettes with 70 % (v/v)alcohol then with Lookout DNA erase solution; keep on a clean surface throughout manipulation.
Crush enough ice to fill the PCR box or a beaker bowl till it's 3/4 filled.
Remove all reagents needed from -20 °C and 4 °C and thaw on ice
Prepare a sterile labelled 1.5 mL microcentrifuge tubes or larger volume tubes depending on amount of PCR mix needed, and leave open on ice.

SECTION: Preparation of 1x PCR Master mix formulations
5. Pipetting components to make up the "Wet" mix

Pipette the corresponding amount of PCR grade water to reconstitute the enzyme as was determined during preparation (in this case 30 uL PCR water is used to reconstitute the enzyme) and place on ice.
Pipette all reagents in the tube following the order stated in the table below:

 

 
   OrderComponentsWet Formulation (20% Trehalose)1PCR grade H2O8.8 ul2dNTPs mix (25 mM)0.6 ul310x Thermopol Buffer2 ul4MgSO4 (100mM)0.6 ul5Cellular reagents (Freshly prepared and heat treated)3ul620% (w/v) Trehalose stock solution4.5 ul7*Bromophenol Blue 0.25% (w/v)Azorubine 0.25% (w/v)0.5 ulTotal20 ul

Determine the total volume needed and make necessary calculations to adjust the amount of each component to be pipetted.
Mix well by gentle agitations (avoid using vortex as the mixture will foam)

 

Aliquoting to make a "Dry" mix

Carefully pipette 25 µLof Mix formulation into each 0.2ml PCR tubes
Place PCR tubes (left Opened) in an Airtight container filled halfway with silica beads. 
Place the container in an incubator and dry at37 °C 
Store dried down tubes at4 °C in air-tied sachet or containers filled halfway with silica beads. 



SECTION: Quality control - Functionality testing
6. 
Check the functionality of the produced PCR Master Mix by running control PCR reactions.

We generally use the BenBio internal protocol which can be modified depending on the PCR master mix formulation and concentration being tested.

 


SECTION: Quality control - Functionality testing
7. Running Agarose gel and Visualization

After running the PCR reaction, the samples are visualized to check for amplification as follows:

Prepare 1.5% agarose gel and run the gel electrophoresis to completion.
Visualize to check for DNA bands which signify amplification to show that the enzyme or PCR master mix are functional (able to amplify a specific region of a test DNA template).
SECTION: Preparation of 1x PCR Master mix formulations
4. Preparing reagent stocks

20 Mass / % volume Trehalose (10 mL)

Weigh 2 g of trehalose powder into a clean 50 mL capacity beaker.
Measure 10 mL of distilled water and pour into the beaker to dissolve the powder.
Filter the trehalose stock solution obtained through a 0.22 uM syringe filter in a sterile 15 mL eppendorf tube.
Store at 4 °C for up to 12 months.

0.25 Mass / % volume Azorubine and 0.25 Mass / % volume Bromophenol blue (10mL)

Use a weighing balance to accurately 0.025 g of Bromophenol blue or Azorubine dye into a 15 mL centrifuge tube.
From a 20 % trehalose stock, aliquot some volume into the Eppendorf tube to dissolve the dye powder, to make a 10 mL (dye) solution.
Cork tightly the eppendorf tube and mix the content gently to homogenize (vortex can be used).
Label the tube and either store it at room temperature (away from light) or 4 °C for up to 12 months.