DNA/RNA kits

TECHNICAL CHARACTERISTICS OF HELLABIO DNA ELECTROPHORESIS KITS 

*TBE=Tris-borate-EDTA.

**Add100μl/100mlAgarose
 

Preparation of the Agarose gel:

a) Prepare the working buffer solution by adding 2.5ml of the 40x concentrated TBE buffer in 97.5ml dest. water to get 100 ml  working buffer solution.

b) To get the desired Agarose concentration mixed:

c) Microwave to get complete solution. Leave it to cool on the bench for 5 minutes down to about 60°C. The reason for allowing the Agarose to cool a little before this step is to minimize production of ethidium bromide vapor (is very carcinogenic).

d) Add 100µl (=50ng/100μl) of ethidium bromide to 100 ml Agarose solution and swirl to mix (dispose the contaminated tip into a dedicated ethidium bromide waste container (potassium permanganate solution for 5 minutes).

e) Pour the gel slowly into the tank. Push any bubbles away to the side using a disposable tip; insert the probable comb and leave to set for at least 30 minutes, preferably 1 hour.

f) Pour working buffer solution into the electrophoresis tank to submerge the gel to 2–3 mm depth.

g) Prepare the samples and load according to your protocol.

h) Close the gel tank, switch on the power-source and run the gel at 5 V/cm (if the electrodes are 10 cm apart then run at 5X10= 50 V).

Additional Reagents and Equipments which can be provided by Hellabio:

• Submarine electrophoresis Tank.
• Power supply.

Limitation / Caution:

• Do not use the Agarose gel if it seems to be dried.
• Do not freeze the Agarose gel.
• Store the kit in horizontal position.

TROUBLESHOOTING

If you see faint or no bands on the gel:

• There was insufficient quantity or concentration of DNA.
• The DNA was degraded. Avoid nuclease contamination.
• The DNA was electrophoresed off the gel.
• Improper  light source was used for visualization of ethidium bromide-stained DNA.

If you see smeared DNA bands:

• The DNA was degraded. Avoid nuclease contamination.
• Too much DNA was loaded on the gel.
• Improper electrophoresis conditions were used. Do not allow voltage to exceed ~20 V/cm. Maintain a temperature <30° C during electrophoresis. Check that the electrophoresis buffer used had sufficient buffer capacity. This is done by checking the pH in the anode and cathode chambers.
• There was too much salt in the DNA.
• The DNA was contaminated with protein.

If you see anomalies DNA band migration:

• Improper electrophoresis conditions were used. Do not allow voltage to exceed ~20 V/cm. Maintain a temperature <30° C during electrophoresis. Check that the electrophoresis buffer used had sufficient buffer capacity.
• The DNA was denatured. Do not heat standards [except for l DNA/Hind III fragments (figure 1)] prior to electrophoresis. Dilute DNA standards in buffer with 20 mM NaCl.