TITLE: Functionality test (TBE  electrophoresis buffer)
AUTHORS: Cordellia Cordellia Fulai, Nadine   Mowoh, Stephane  Fadanka

[DESCRIPTION]
This protocol describes the comparative analysis of resolution of bands and band patterns after electrophoresis with the BenBio TBE  buffer formulation as compared with a commercial TBE buffer. We use 0.5 kb and 1kb PCR amplicons loaded on 1.5% (w/v) agarose gel, run electrophoresis at 80V and visualized on a Blue light transilluminator or UV transilluminator.
Our formulation of TBE buffer is able to allow us obtain a higher resolution of smaller DNA fragments on agarose gels.


[BEFORE_START]
Ensure the buffer sachet is not broken and powder remains free from moisture to enable the resulting buffer to stay longer and avoid compromising the gel results.

[GUIDELINES]
This protocol describes the steps in testing the functionality of TBE buffer 

[STEPS]
SECTION: PCR amplification
1. In order to have the PCR amplicons to use for this experiment we typically amplify the 0.5 and/or 1kb regions of the Lambda DNA in a PCR reaction as follow:

Preparing for PCR amplification of 0.5 and 1kb Lambda DNA

Crush some ice and put in a clean bowl
Remove and thaw all PCR reagents on ice (dNTP mix, 10x PCR buffers, primers, DNA template, DNA polymerase, PCR water)
Label 3 or more 0.2 mL PCR tubes (E.g with the name of the DNA template and a negative control tube) and place them on ice as shown in figure 1.

 

 
SECTION: Functionality test of TBE buffer
4. 
Preparing 1x TBE buffer from buffer powder sachet

 

To make a 1x Buffer - 1 L

Open up a sachet of 10x TBE buffer (containing 85.14 g of buffer powder) and pour into a 1 L capacity beaker.
Measure 0.5 L of distilled water using a measuring cylinder into the beaker and stir until it the powder completely dissolves. You may add a magnetic flea and use a magnetic stirrer to ease dissolution of the powder into a clear liquid which remains stable and clear for up to 1 year at room temperature.
Measure out 100ml of the 10x buffer into another 1 L capacity beaker
Measure 900ml of distilled water and add into the beaker to make up the volume to 1 L. The 1x TBE buffer is stored at room temperature for subsequent use. 

SECTION: Functionality test of TBE buffer
5. Preparing 1.5 % TBE agarose gel 

 

 

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).

SECTION: Functionality test of TBE buffer
6. Casting and loading the Electrophoresis 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).
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 1).
Pipette 1 part of the 6x DNA loading dye and 5 parts of the negative control, mix and load into the second well.
Pipette 1 part of the 6x DNA loading dye and 5 parts of the DNA amplicons, mix and load into the subsequent wells (depending on the number of amplicons) (Figure 2).
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 20 minutes run).
    
SECTION: Functionality test of TBE buffer
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 of the DNA and visibility of the DNA bands of the BenBio TBE buffer with commercial TBE buffer to show that the BenBio TBE buffer does not negatively affect the migration of DNA nor hinder visualization of the DNA bands on the gel.

SECTION: PCR amplification
2. Assemble the reaction mix into 20 ul volume in 0.2 mL PCR tubes in the order shown in figure 2 below.
 
 
 

Hold the tubes up and gently mix by tapping tubes and place in a thermocycler.
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.
 
3. Thermocycling