TITLE: Functionality test (10x PCR buffer)
AUTHORS: Nadine    Mowoh, Stephane  Fadanka

[DESCRIPTION]
In this protocol we describe how to test the functionality of the BenBio 10x PCR buffer by showing that the buffer is able to provide a suitable condition for DNA polymerase to amplify a DNA template in a PCR reaction when compared to a commercial PCR buffer. We confirm the result by running agarose gel electrophoresis of the resulting DNA amplicon.

The BenBio 10x PCR buffer provides optimum PCR conditions for OpenVent in PCR amplification reactions.

[BEFORE_START]
Make sure all PCR reaction components are available.
Prepare an ice bowl because all pipetting will be done on ice.

[GUIDELINES]
This protocol describes the steps in testing the functionality of our 10x PCR buffer formulation.

[STEPS]
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2. Mixing

Hold the tubes up and gently flick the tubes to mix the components and place the tubes in a thermocycler.
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3. Thermocycling

Input the cycling parameters as indicated in the table below and run. (The amplification/running time will depend on the size of the DNA template).
 
     ABCD1Phase cycleTemperature (°C)Duration (sec)Number of cycles2Initial denaturation951203Denaturation95304Annealing50 to 65 (depending on primers)2030 to 355Extension72variable based on primers6Final extension72120

After amplification, we confirm the functionality of our PCR buffer by running a 1.5% agarose gel.
 
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4.  Performing Agarose gel electrophoresis
Prepare 10x and 1x TBE buffer
We  use this internal protocol to prepare a 10x and subsequently a 1x TBE buffer which will be used in preparing the agarose gel and running buffer.

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5. Preparing 1.5 % agarose gel for electrophoresis

 

 

Use a weighing balance to weigh out 0.375 g of agarose powder and pour into a 150 mL or any appropriate size glass beaker.
Use a measuring cylinder  to measure 25 mL of 1x TBE buffer and add to the agarose powder in the beaker (The volume of gel you will need to make will depend on the size of the casting tray.)
Swirl the beaker for about 5 seconds to dissolve the powder
Put the beaker into the Microwave and heat at medium high for 1 minute until it boils
Remove the molten agar from the Microwave and allow to cool for 30 seconds (about when you can comfortably keep your hand on the beaker).
Add EtBr gel stain to a final concentration of 0.5 μg/mL (usually about 2.5 µL to 3 µL of lab stock solution per 25 mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light or Blue light.
Swirl the beaker gently to mix and distribute the DNA gel stain (swirl gently to avoid bubbles).
Pour the molten agar into the gel casting tray with the well combs in place (Pour slowly to avoid bubbles which will disrupt the gel),  allow to solidify for about 15 to 20 minutes as shown in Figure 2 below.

   
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6. Casting and loading the Electrophoresis gel

After the gel is solidified, remove the comb and place the casting tray into the gel box or electrophoresis unit.
Fill the electrophoresis unit with 1x TBE buffer to cover the gel (the amount of running buffer to use will depend on the size of the electrophoresis unit).
Pipette 3 μLof DNA ladder (a collection of DNA fragments of known lengths that helps you determine the approximate length of a DNA fragment by running it on an agarose gel alongside the DNA amplicon), into the first well (Figure 3). ( The DNA ladder is loaded without mixing with a loading dye because it contains a tracking dye already)
Pipette 1 part of the 6x DNA loading dye and 5 parts of the DNA amplicon, mix and load into the next 4 wells (could load more or less wells depending on the number of amplicons) (Figure 4).
Connect the electrophoresis unit to a power or voltage source by connecting the electrodes from the power source to the electrophoresis unit (Black is negative, red is positive). The DNA is negatively charged and will run towards the positive electrode (always run to red). 
Run to finish (we adopt 80 to 100 Volts for 150 minutes run).
    
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7. Visualizing and Analyzing the gel

Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box unto a gel visualization system.
Using any device that has UV light (UV transilluminator), visualize your DNA fragments. The fragments of DNA are usually referred to as ‘bands’ due to their appearance on the gel.
Using the DNA ladder in the first lane as a guide (the manufacturer's instruction will tell you the size of each band), you can infer the size of the DNA in your sample lanes.

After the run, compare the migration pattern and visibility of the DNA bands of the BenBio 10x PCR buffer with that of the commercial 10x PCR buffer to show that the BenBio 10x PCR buffer is able to provide a suitable PCR condition for the amplification of the specific DNA templates. 
SECTION: Functionality test of 10x PCR buffer
1. Preparing PCR reagents and components

Thaw all reagents on ice in a bowl.
Label 2 reaction tubes (0.2 mL PCR tubes) "test buffer" and "control buffer" or more tubes depending on the number of replicates you choose to do.
Pipette the reaction components into the tubes as indicated in the table below (do this while working on ice).