TITLE: Functionality test (OpenVent polymerase, PCR Master Mixes)
AUTHORS: Nadine          Mowoh, Stephane  Fadanka, Shalo  Minette

[DESCRIPTION]
After production, we typically subject our products to a batch of quality control assays to ascertain their functionality, efficacy and ability to meet their intended purpose.

Strict quality control is necessary when producing or using enzymes (whether purified or unpurified) and other molecular biology reagents. 

Quality control for functionality involves incubating the enzymes or PCR master mix in a PCR reaction mixtures and testing for their ability to amplify a specific region of a DNA template and the results recorded in terms of the presence or absence of amplification by bands on an electrophoresis agarose gel.

The Quality control tests are done at specific regular intervals and results documented for follow up. Incase any product fails the quality control test, actions are taken to verify the cause and make sure it does not repeat. 

More than one test may be carried out for a particular product and the quality manager must approve and sign the certificate of analysis (a document that reveals the different quality control tests that have been performed on a reagent) to show that the product passes all the quality control tests assigned for it before a product is released for use.
Quality control of a product is done in two stages - the first involves a batch of tests carried out within the facility where the reagents are produced (Internal quality control) and the second involves testing for functionality by an external facility (External quality control).

In this protocol we describe how to carry out Functionality test of the following:
OpenVent Enzyme
PCR Master Mix - All formulations (wet and dry).


[BEFORE_START]
Clean and disinfect all work surfaces with a 1:10 dilution of bleach followed by 70% alcohol.

[GUIDELINES]
Before carrying out quality control on products research should be carried out on the possible assays that apply for that particular product and availability of resources.

[STEPS]
SECTION: Functionality test - PCR amplification
1. DNA polymerase Enzyme and PCR Master mix

 

Preparing reagents

Thaw all reagents on ice in a bowl
Label reaction tubes (0.2 mL PCR tubes) according to the number of samples, and include controls in each run (negative and positive controls) as needed.

Polymerase enzyme type:

If using a 10x cellular reagent enzyme, rehydrate the enzyme with 30 μL of sterile PCR grade water, flick tube and keep on ice.
If using a pre-purified polymerase or Master mix, remove from the freezer and keep on ice as shown in figure 1 below.
  
SECTION: Functionality test - PCR amplification
3. Thermocycling:

   Initial Denaturation95120 sec       Denature95°C30 secs       Anneal50-65°C(depending on primers)20 sec       Extend72°CVariable based on primersFinal Extension72°C120secs
 

At the end of the thermocycling process, run agarose gel electrophoresis to check for amplification of the DNA template and establish functionality of the enzyme and PCR master mix.
SECTION: Functionality test - PCR amplification
4. Preparing Agarose gel for Electrophoresis

Prepare a 1.5% TBE agarose gel as follows:

Use a weighing balance to weigh 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).

Casting the gel

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.
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).


SECTION: Functionality test - PCR amplification
5. Loading and running the gel wells 

 

Pipette 3 μL of DNA ladder (the size of the ladder would depend on size of the DNA template) into the first well. 
Pipette 1 part of the DNA loading dye and 5 parts the negative control carefully to avoid bubbles and load into the second well.
Pipetting 1 part of the DNA loading dye and 5 parts of DNA amplicons or test samples, mix and load in the subsequent wells. (Do same for the positive control).
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 Volts for 15 minutes run).

SECTION: Functionality test - PCR amplification
2. Pipetting
In each PCR tube, pipette and combine the following PCR reaction components in the order shown in table 1 below (work on ice while pipetting).
 
 


Hold the tubes up and gently flick the tubes to mix the components and place in a thermocycler.
Input the cycling parameters as indicated in the table below and run. 
 

SECTION: Functionality test - PCR amplification
6. Visualization of gel and Interpretation of gel results

The amplification results are visualized by transferring the gel into a UV transilluminator or blue light transilluminators.
With the help of the DNA ladder, determine if the amplification product is of the expected size and the bands are sharp and distinct by comparing with the DNA polymerase enzyme and PCR Master Mix standards.

 


The results obtained will determine if the reagent needs further testing or follow up or would be declared "good to use".