TITLE: Functionality test (Borax Buffer)
AUTHORS: Nadine   Mowoh, Shalo  Minette, Stephane  Fadanka

[DESCRIPTION]
This protocol describes the comparative analysis of visual and band patterns after electrophoresis with the BenBio Borax buffer 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 100 Volts and visualize on a Blue light transilluminator or UV transilluminator.

The Borax buffer has an advantage of allowing the gels to be run at very high voltages (100 V and 150 V) without the buffer heating up and affecting the gel and also faster reading of gel results within 10 to 15 minutes. 
Contrary to the Borax buffer, TBE buffer gels are run at 80 V to 90 V to avoid heating up the buffer and it takes up to 20 to 30 minutes to read gel results.

[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 preparing and testing the functionality of our formulation of Borax buffer

[STEPS]
SECTION: PCR amplification
1. Preparing PCR amplicons

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 follows:

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 1below.
 

 

SECTION: Functionality test of Borax buffer
4. Preparing 1x Borax buffer

 

To make a 1x Borax buffer - 1 L

Open up a sachet of 10x Borax buffer (containing 10 g buffer powder) and pour into a 2 L capacity beaker.
Measure 1 L of 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.
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 Borax buffer is stored at room temperature and is stable for up to 1 year.

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

 

 

Prepare a 1.5 % Borax agarose gel for electrophoresis 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 Borax 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 Borax 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 Borax 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 2).
Pipette 1 part of the 6x DNA loading dye and 5 parts of the DNA amplicon, mix and load into the wells (depending on the number of amplicons) (Figure 3).
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 100 to 200 Volts for 15 minutes run).
    
SECTION: Functionality test of Borax 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 Borax buffer with TBE buffer to show that the Borax buffer does not negatively affect the migration of DNA nor hinder visualization of the DNA bands on the gel.



SECTION: PCR amplification
2. Pipetting
Assemble the reaction mix into 20 ul volume in 0.2 mL PCR tubes in the order shown in table 1 below.

    ABC1ComponentAmount in ul -in each 0.2 mL tubeNegative Control tube2PCR watervariable up to 20ulvariable up to 20ul3Thermopol buffer 10x224dNTP Mix 10 mM0.40.45Fw/Rv primers1/11/16DNA template0.5 (depending on the concentration)/7Enzyme2 units (1ul)2 units (1ul)


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.
SECTION: PCR amplification
3. 
Thermocycling




     ABCD1Phase cycleTemperature (°C)Duration (sec)Number of cycles2Initial denaturation951203Denaturation95304Annealing50 to 65 (depending on primers)2030 to 355Extension72variable based on primers6Final extension72120