Jun 29, 2022

Public workspaceConcentrated DNA Loading dye stock: User protocol

This protocol is a draft, published without a DOI.
  • 1Beneficial Bio, Mboalab
avatar.png
Icon indicating open access to content
QR code linking to this content
Protocol Citation: Stephane Fadanka, Nadine Mowoh 2022. Concentrated DNA Loading dye stock: User protocol. protocols.io https://protocols.io/view/concentrated-dna-loading-dye-stock-user-protocol-cbtvsnn6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: June 22, 2022
Last Modified: June 29, 2022
Protocol Integer ID: 65109
Abstract
Beneficial Bio 6X DNA Loading Dye is a premixed loading buffer used to prepare samples for
loading onto agarose and polyacrylamide gels. It contains bromophenol blue and xylene
cyanol dyes for visual tracking of DNA during electrophoresis. The trehalose ensures that
DNA forms a layer at the bottom of the well and facilitates sample loading. The EDTA
chelates metal ions and stops metal-dependent enzymatic reactions. The solution contains
SDS which results in sharper bands by removing restriction enzymes from DNA.

Features:

  • Double tracking: Xylene cyanol, Bromophenol blue
  • No interaction with bands during gel exposure to UV or Blue light
  • Inhibits metal-dependent nucleases (EDTA)

Applications:

Preparation of DNA ladders, markers, and samples for loading on agarose or polyacrylamide
DNA electrophoresis.
Guidelines
Follow good laboratory practices when carrying out experiment although all reagents used are generally safe.
Materials
  • Concentrated DNA loading dye stock
  • 20% Trehalose
  • DNA samples
  • Micro Pipette and tips
  • Agarose gel electrophoresis system
  • Gel visualization system
Before start
Ensure all materials, equipment and chemicals to be used for this experiment are all in place before starting.
User Protocol
User Protocol

Note
Our DNA loading dye stock is usually prepared in a concentrated form that is stored in the fridge and diluted to 6x working stock before it is mixed with DNA for loading.

Preparing 1ml of 6x working stock:

  1. Vortex or shake the concentrated stock tube for 10 seconds prior to use.
  2. Dilute 1 part of concentrated DNA Loading Dye stock with 9 parts of 20% Trehalose (pipette Amount100 µL from the concentrated dye stock and add Amount900 µL of Concentration20 % (v/v) Trehalose to make a 1ml 6x working stock).
  3. Cork the tube firmly and mix by inverting the tube several times.
  4. Store the working solution at room temperature orTemperature4 °C for up to 12months, for longer periods, store atTemperature-20 °C .
  5. To use, dilute the nucleic acid solutions 1:5 to give a 1x solution e.g. add Amount1 µL 6x DNA loading dye for everyAmount5 µL DNA.
  6. Load the sample into a precast agarose gel well, run to finish and visualize the gel with the help of a UV transilluminator or blue gel system.



Notes and troubleshooting
Notes and troubleshooting
  • BenBio 6X DNA Loading Dye enables easy tracking and sample migration and follow up during the run without creating background noise during visualization.
  • If you notice any interference of the tracking dye with the DNA bands, check the working concentration and try further dilutions to find the one that works for you.

Note
Also the storage temperature is important as the trehalose in the dye might loose its efficacy over time as noticed if not stored properly, hence DNA loading dye may not be able to cause samples to sink into the gel wells (may cause the samples to leak or just spread out instead).