TITLE: Beneficial Bio Products: Quality control tests 
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 involves incubating the enzymes or reagents in reconstituted recombination assays to eliminate the possibility of relevant protein or nucleic acid contaminants that may interfere with recombination assays and lead to misinterpretation of experimental data. Here batch by batch quality controls are done for each new product before it is released to the market. 

The Quality control tests are done at specific regular intervals and results documented for follow up and action in case any product fails the quality control test. 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.
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).


[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 tests
1. 
Pipetting:
 

DNA polymerase and Master mix enzyme

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 tests
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 template DNA.
 
SECTION: Functionality tests
3. Preparing Agarose gel for Electrophoresis
Follow this protocol to prepare and cast the gel.
SECTION: Functionality tests
4. Loading the gel wells and Visualisation of gel

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

The amplification results are visualized by transferring the gel into a UV transilluminator or blue gel transilluminator.

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, PCR buffer, Agarose gel electrophoresis buffer, DNA gel stain and DNA loading dye standards.
SECTION: Nuclease activity
5. 
 

SECTION: Nuclease activity
6. Pipetting
Pipette reaction mixtures in the order described below and incubate as indicated:

 
Checking and interpreting results:
After incubation period the reaction is stopped, and an 1.5% agarose gel prepared, run and visualized as described in steps 3 and 4 above.

Any change in band intensity and migration position of the plasmid DNA bands compared to the negative control signals potential endonuclease activity. Linear DNA should run true to size(known size of plasmid used), supercoiled DNA should run faster and relaxed circle DNA should run slower(a single strand cut).
SECTION: Visualization of low molecular weight DNA and low DNA concentration
7. 
 

Follow the steps in preparing a 2% agarose gel as described in step 3 above and load by pipetting 1part of the DNA loading dye and 5 parts of amplicon or test sample . DNA templates or amplicon in this case  is a 50 or 100bp DNA ladder therefore it can be loaded without loading dye needing 3-5ul of the ladder. Load directly into the gel well in replicates.
Run gel to finish and visualize the resulting band separation to confirm separation of the ladder into visible distinct bands (from the lowest to highest base pair bands) using a UV transilluminator or blue gel system.

To confirm a low DNA concentration detection/visualization property of the product:
Make 1:5 serial dilutions of the 100bp DNA ladder or any DNA amplicon and use the different dilutions as amplicons to load a gel well.
Allow the gel to run through and visualize to determine the lowest concentration detectable by the gel stain.

SECTION: Physical property- Stability and pH
8. 
 

To confirm the weight of the powder buffer sachet:

Weigh the individual powder components that constitute the buffer ( the individual powders could be pre-dried if necessary in an incubator with silica gel beads at 37c)
Put them together in a beaker and mix 
Pour the powder mix in a zip lock sachet and store in an air tight container for 1 to 3 days.
After, open the sachets and pour the powder into a weighing boat and measure the weight of the powder
The weight should be approximately close to or equal to the weight of the powder 3 days before.

SECTION: Physical property- Stability and pH
9. To confirm the pH of the buffer solution:
Pour the powder buffer into an appropriate size beaker and add the appropriate amount of distilled water as indicated on the powder sachet (e.g 1 sachet in 1L water).
Mix until a clear solution is obtained
Use a pH meter to check the pH of the solution which should be between 8.3 and 8.5.