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

[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 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 he 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
1. DNA polymerase Enzyme and PCR Master mix

 

Pipetting:

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

Polymerase enzyme type:

If using a 10x cellular reagent enzyme- Rehydrate the enzyme with 30μlof 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.

In each PCR tube, pipette and combine the following reagent components following the order in the table below (work on ice while pipetting).

 

 


SECTION: Functionality test
2. Thermocycling:

Connect a Thermocycler and program the protocol to run following the thermocycling steps listed below. The exact annealing temperature will depend on the choice of DNA template and primers used.

 
   Initial Denaturation95120 sec       Denature95°C30 secs       Anneal50-65°C(depending on primers)20 sec       Extend72°CVariable based on primersFinal Extension72°C120secs
The time of run will depend on the size of the DNA template and number of cycles of amplification (usually we use do 35 to 40 cycles for 0.5kb Lambda-50ng/ul).
At the end of the thermocycling process, run agarose gel electrophoresis to check for amplification of the DNA template.
 
SECTION: Functionality test
3. Preparing Agarose gel for Electrophoresis
Follow this protocol to prepare and cast the gel.
SECTION: Functionality test
4. Loading the gel wells 

Pipette 3 to 5μl of DNA ladder (size depending on size of the DNA template) unto the first well then pipette the negative control carefully to avoid bubbles and load into the second well.

Follow the step in loading by pipetting 1part of the DNA loading dye and 5 parts of amplicon or test sample, mix your amplicons with DNA loading dye and load in the subsequent wells (in the case of the master mixes, they are loaded directly as they already contain the tracking dye and components to help them sink in the well).

Allow the gel to run for 15-30mins at 48V on 0.8, .5% or 2% agarose gel in TBE buffer.

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 looking exactly like those from the  DNA polymerase enzyme and PCR Master Mix standards.