Nov 15, 2015
Agarose gel electrophoresis, 1.2% with Ethidum bromide
Forked from Gel Electrophoresis
Protocol Citation: Harold Bien: Agarose gel electrophoresis, 1.2% with Ethidum bromide. protocols.io https://dx.doi.org/10.17504/protocols.io.ds76hm
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
Created: September 11, 2015
Last Modified: March 27, 2018
Protocol Integer ID: 1599
Abstract
Separates molecules based on size. Great for checking DNA after a Restriction Digest. This protocol is for a 1.2% agarose gel, sufficient for resolving DNA 400bp to 7kb (conservatively). The gel electrophoresis system is a 7.5cm x 10cm gel bed area with approximately 15cm electrode distance. The minimum volume required for a 7.5cm x 10cm x 0.5cm gel is 37.5mL. The maximum gel height for 50mL is 0.67cm.
Materials
MATERIALS
1 kb Plus DNA Ladder - 200 gel lanesNew England BiolabsCatalog #N3200S
Gel Loading Dye, Purple (6X), no SDS - 4.0 mlNew England BiolabsCatalog #B7025S
GeneMate LE Quick Dissolve Agarose, 500gBioexpressCatalog #E-3119-500
Ethidium bromide, 10mg/mL, 10mLAmrescoCatalog #X328-10ML
TAE (TRIS-ACETATE-EDTA) Buffer, 50xAmrescoCatalog #K915
Horizontal mini-gel kitC.b.s ScientificCatalog #MHU-202
STEP MATERIALS
GeneMate LE Quick Dissolve Agarose, 500gBioexpressCatalog #E-3119-500
TAE (Tris-Acetate-EDTA) buffer, 1x
Ethidium bromide, 10mg/mL, 10mLAmrescoCatalog #X328-10ML
Gel Loading Dye Blue (6X) - 4.0 mlNew England BiolabsCatalog #B7021S
GeneMate LE Quick Dissolve Agarose, 500gBioexpressCatalog #E-3119-500
TAE (Tris-Acetate-EDTA) buffer, 1x
Ethidium bromide, 10mg/mL, 10mLAmrescoCatalog #X328-10ML
Gel Loading Dye Blue (6X) - 4.0 mlNew England BiolabsCatalog #B7021S
Protocol materials
Ethidium bromide, 10mg/mL, 10mLAmrescoCatalog #X328-10ML
Horizontal mini-gel kitC.b.s ScientificCatalog #MHU-202
TAE (Tris-Acetate-EDTA) buffer, 1x
1 kb Plus DNA Ladder - 200 gel lanesNew England BiolabsCatalog #N3200S
Gel Loading Dye, Purple (6X), no SDS - 4.0 mlNew England BiolabsCatalog #B7025S
TAE (Tris-Acetate-EDTA) buffer, 1x
Gel Loading Dye Blue (6X) - 4.0 mlNew England BiolabsCatalog #B7021S
GeneMate LE Quick Dissolve Agarose, 500gBioexpressCatalog #E-3119-500
Ethidium bromide, 10mg/mL, 10mLAmrescoCatalog #X328-10ML
Gel Loading Dye Blue (6X) - 4.0 mlNew England BiolabsCatalog #B7021S
GeneMate LE Quick Dissolve Agarose, 500gBioexpressCatalog #E-3119-500
GeneMate LE Quick Dissolve Agarose, 500gBioexpressCatalog #E-3119-500
Ethidium bromide, 10mg/mL, 10mLAmrescoCatalog #X328-10ML
TAE (TRIS-ACETATE-EDTA) Buffer, 50xAmrescoCatalog #K915
GeneMate LE Quick Dissolve Agarose, 500gBioexpressCatalog #E-3119-500
TAE (Tris-Acetate-EDTA) buffer, 1x
Ethidium bromide, 10mg/mL, 10mLAmrescoCatalog #X328-10ML
Gel Loading Dye Blue (6X) - 4.0 mlNew England BiolabsCatalog #B7021S
Safety warnings
Ethidium Bromide potentially acts as a mutagen or carcinogen.
Before start
Have a DNA Sample ready, typically either from PCR or a recently performed Restriction Digest. Dilute down the 50X TAE Buffer to 1X using sterile filtered water. For the dual-sided 1.0mm gel comb MHC-10-0816 the maximum volume is 35/13μL. For the dual-sided 1.5mm gel comb MHC-15-1014, the maximum sample volume is 38/25μL.
Prep Work
Prep Work
Weigh out 0.6 g (1.2% w/v of 50mL) agarose and add it to the Erlenmeyer Flask.
1 g
GeneMate LE Quick Dissolve Agarose, 500gBioexpressCatalog #E-3119-500
Add 50mL of 1x TAE buffer
50 mL
TAE (Tris-Acetate-EDTA) buffer, 1x
Place Erlenmeyer Flask in microwave. Set to wait 30 seconds, then full power (P10, 1250W) for 20 seconds followed by low power (P1, 125W) for 30 seconds or until solution is clear and agarose is completely dissolved.
00:00:50
Note
Press 'Timer', enter 30 seconds. Then press 'Power', enter 20 seconds. Then press 'Power' 10 times to select P1, then enter 20 seconds. Press 'START'.
Remove Erlenmeyer Flask from microwave and let it sit on the lab bench to cool just until you can comfortably pick it up.
00:03:00
Add 1μL concentrated ethidium bromide (10mg/mL) into the flask and swirl to mix, taking care not to introduce bubbles.
1 µL
Note
Final concentration of ethidium bromide will be 10μg/50mL or 0.2μg/mL
Ethidium bromide, 10mg/mL, 10mLAmrescoCatalog #X328-10ML
Place gel tray on clamp and clamp securely. Add well plates where you want wells and use a level to ensure it is balanced.
Note
For MHC-10-0816 1mm thick dual-sided 8/16 well gel combs, the maximum recommended sample volume is 35μL for 8 wells and 13μL for 16 wells.
For MHC-10-1014 1mm thick dual-sided 10/14 well gel combs, the maximum recommended sample volume is 26μL for 10 wells and 17μL for 14 wells.
For MHC-15-0816 1.5mm thick dual-sided 8/16 well gel combs, the maximum recommended sample volume is 52μL for 8 lanes and 19μL for 16 lanes.
For MHC-15-1014 1.5mm thick dual-sided 10/14 well gel combs, the maximum recommended sample volume is 38μL for 10 lanes and 25μL for 14 lanes.
Pour contents of the Erlemeyer Flask into the gel tray and let it sit for 30 minutes, or until a blue tint appears.
00:30:00
Loading the Gel
Loading the Gel
Remove the well plates carefully as to not tear the gel and remove the tray from the clamp, but ensure the gel remains in the tray.
Place gel tray into gel electrophoresis apparatus with the wells closer to the negative/black end.
Pour additional TAE Buffer to fill each side of the apparatus and to create a thin layer of buffer covering the top of the gel.
Prepare DNA ladder and samples by adding 6x blue dye
Note
Recommend diluting DNA ladder 1:10 in sterile filtered water and loading 200ng of DNA ladder for the 1.5mm thick 14-well lane (3.5mm wide).
Gel Loading Dye Blue (6X) - 4.0 mlNew England BiolabsCatalog #B7021S
Pipette your samples into each well.
Note
For the 1.5mm gel comb MHC-15-1014, recommended DNA mass is 200-500ng for 14-well.
Running the Gel
Running the Gel
Place lid on apparatus and plug cables into high voltage power supply. Run at 100V (~6.6V/cm) for 45-60 minutes or until the loading dye has sufficiently migrated down the gel.
00:45:00
Note
Ensure that the negative terminal (typically black) is plugged into the negative/black terminal on the power supply and the loading well are closest to the black/negative side.
Visualizing DNA bands
Visualizing DNA bands
Gel can be imaged on UV transilluminator through the UV-transparent gel tray or removed and wrapped in plastic wrap for storage at 4°C for later use.