Protocol for Agarose Gel Electrophoresis of PCR Products

Date approved:

Approved by:

Introduction

Agarose gel electrophoresis separates DNA molecules by size. DNA is negatively charged and will pass through the gel matrix towards the positive electrode when subjected to an electric field. Short molecules will move more quickly than larger ones and will migrate further through the gel. The gel contains SYBR Safe DNA gel stain so that the DNA can be visualised using UV light. A size marker is added to the gel so that the size and strength of the PCR product can be confirmed.

Materials

Labcoat, safety spectacles and disposable nitrile gloves

Agarose gel with SYBR Safe stain

Electrophoresis tank of appropriate size

1L Measuring Cylinder

10xTBE Buffer (Invitrogen – Part Number 15581-044)

P20 Gilson Pipette

Gilson D200 diamond tips (Anachem, Part Number F161930)

Gilson Repeater pipette

Distritip Micro 125µl (aliquots 1 – 12.5µl) (Anachem, Part Number F164100)

Anachem Octapipette

Microtitre plate(Premier Scientific, Part Number 29100)

5x Gel Loading Buffer

Size Marker ФX174DNAHaeIII (Thermo Scientific Abgene, Part NoAB-0389)

PCR product

Power Pack

UV Transilluminator

Safety Notes

Read and sign the COSHH risk assessment, Task risk assessment and SOP for ‘Electrophoresis of an Agarose Gel with SYBR Safe DNA Gel Stain’, before beginning the procedure.

The laboratory rules must be adhered to at all times.

Fastened up lab coat, safety goggles and disposable nitrile gloves must be worn.

Do not open laboratory doors, use computers or the telephone with gloves on.

Know how to dispose of waste before beginning the procedure. Follow the laboratory disposal protocol.

Extreme care!Ultra Violet Light may cause serious burns to the skin, may cause genetic damage leading to skin cancer and may cause serious eye damage. Ensure that the transilluminator is housed inside the safety cabinet and that the door of the cabinet is securely closed before the UV light is turned on.

Electrical hazard! Never use electrical equipment with wet gloves on. Keep water and buffer away from all electrical connections. Do not use an electrical connection if it is wet.

Procedure for Agarose Gel Electrophoresis with SYBR Safe DNA gel Stain

  1. Using a 1L measuring cylinder, measure 100mls of 10xTBE. Make up to the 1L mark with MilliQ water. Cover the top with parafilm and holding over the sink, invert 3 times to mix.
  2. Gently pour the 1xTBE into the electrophoresis tank until the middle plateau is just covered by buffer. Caution! Pour buffer into the tank from the corner opposite the electrodes – do not allow the electrodes to become wet.
  3. Remove the combs from the set gel by gripping firmly between finger and thumb and pulling straight up. Set each comb down gently with the teeth facing upwards. Caution! Take care not to touch the comb teeth.
  4. Loosen the screws of the template holder until the gel casting tray can be removed. Transfer the tray containing the gel into the electrophoresis tank making sure that the wells are placed nearest to the negative electrode. NB!The negative electrode is black and the positive electrode is red. Top up the level of 1xTBE buffer until it just covers the surface of the gel (too much buffer will cause the gel to run less quickly. The current will flow through the route of least resistance and travel through the buffer).
  5. Prepare the size marker (one for each row of wells): aliquot 2µl of 5xGel Loading Buffer, add 1µl of ФX174DNAHaeIII, add 7µl MilliQ water. Mix by pipetting up and down 3 times. Add to the outside left hand well of each row.
  6. Spin down the PCR product in the Sigma 4-15C plate spinning centrifuge at 1500 rpm for 30 seconds.
  7. Using a Gilson repeater pipette with micro tip, aliquot 2µl of 5x gel loading buffer per sample into a microtitre plate.
  8. Using a either a Gilson P20 pipette or an octapipette (depending on how many samples are to be loaded), add 1µl* of PCR product to the 5xGLB. (*The volume of PCR product to run will depend on the size of the reaction. For example, for a 5µl PCR reaction run 1µl on the gel, for a 25µl reaction add 5µl PCR product to the gel). Mix by pipetting up and down 3 times. Load onto the gel in a known order.
  9. Place the lid onto the electrophoresis tank and connect the leads to the electrodes. The black lead connects to the black (negative) electrode and the red lead connects to the red (positive) electrode. Following the same principleconnect the leads to the power pack. Plug the power pack in and turn it on. Set the voltage at 100-120V and press the ‘run’ button. Confirm that the current is running by checking at the electrodes for the formation of bubbles of gas. Allow the gel to run for 30-40 minutes or until sufficient separation is achieved.
  10. Stop the power pack and turn it off. Disconnect the leads from the power pack and the electrophoresis tank.
  11. Bring the metal carrying tray (stored on the shelf underneath the lab sink) onto the bench right beside the electrophoresis tank. Line the metal tray with tissue paper from the white roll.
  12. Remove the lid of the electrophoresis tank and lift the gel casting tray containing the gel(Take care not to tilt the casting tray - the gel is very slippery and can easilyslide and fall onto the floor!). Carefully transfer into the metal tray lined with tissue paper. Carry the metal tray to the transilluminator and set it down on the bench at the right hand side of the safety cabinet.
  13. Remove one glove and with the ungloved hand open the cabinet door.
  14. With the gloved hand, place the gel casting tray containing the gel onto the centre of the transilluminator.
  15. With the ungloved hand close the cabinet door securely.
  16. Continue by following the Protocol ‘Capturing an image of a Gel on the UV transilluminator’.