TITLE: E-Gel™ 48-Well 2% Agarose Gels  
AUTHORS: Allyson Hirsch, George  Testo

[DESCRIPTION]
E-Gel™48 Gels are pre-cast, ready-to-use, 48-well agarose gels designed for medium-throughput resolution of DNA fragments. Each gel contains 48 sample wells and 4 marker wells in a 2% high-resolution agarose. These types of standardized gels can be ran on an E-Base platform and visualized using Gel Doc XR+. 

[BEFORE_START]
Use 20−100 ng DNA per band for samples containing one unique band or up to 500 ng per lane for samples containing multiple bands. 

Prepare DNA samples in a total sample volume of 15 μL for E-Gel® 48 Gels in deionized water or loading buffer (we recommend using a final loading buffer concentration of 10 mM Tris-HCl; 1 mM EDTA, pH 7.5; 0.005% bromophenol blue; and 0.005% xylene cyanol FF). 

Dilute samples containing >50 mM NaCl, >100 mM KCl, >10 mM acetate ions, >10 mM EDTA (high salt samples) 2- to 20-fold in deionized water, TE, or loading buffer, in a final volume of 15 μL. 

[STEPS]
SECTION: Preparing Loading Plate(s)
1. DNA Away and Ethanol the workstation.
SECTION: Preparing Loading Plate(s)
2. Thaw and spin down samples.
SECTION: Preparing Loading Plate(s)
3. Add 13 µL of molecular grade water to the first 48 wells of a 96-well skirted plate.

SECTION: Preparing Loading Plate(s)
4. Set multichannel pipette to 35 µL (more than 30uL in each PCR plate well). 

SECTION: Preparing Loading Plate(s)
4.1. Combine A, B, C into B and E, F, G into F.

 

SECTION: Preparing Loading Plate(s)
5. Spin down PCR plate (combined) with a temporary seal.
SECTION: Preparing Loading Plate(s)
6. Add 2 µL from B, D, F, H (sample and NTC) to skirted plate with water (do this for each PCR sample or plate).

Combine B into A

Combine D into B

Combine F into C

Combine H into D

SECTION: Loading E-Gel 48-Well 2% Agarose Gels
8. Load the EGel:
SECTION: Loading E-Gel 48-Well 2% Agarose Gels
8.1. 15 uL sample/water dilution mix to odd rows (top and bottom)
SECTION: Loading E-Gel 48-Well 2% Agarose Gels
8.2. 15 uL NTC/water dilution to even rows (top and bottom)
SECTION: Loading E-Gel 48-Well 2% Agarose Gels
8.3. 15 uL ladder dilution to each of the wells marked “M” in each of the 4 corners
SECTION: Running E-Gel 48-Well 2% Agarose Gels
9. Run EGel in the E-Base Invitrogen for 18 min.

SECTION: Imaging E-Gel 48-Well 2% Agarose Gels
10. Take EGel to a Gel Imaging System: Gel Doc XR+ System
SECTION: Imaging E-Gel 48-Well 2% Agarose Gels
10.1. Turn on EpiWhite, load EGel into the bottom tray and close.
SECTION: Imaging E-Gel 48-Well 2% Agarose Gels
10.2. Open computer program and select File - Gel Doc XR.
SECTION: Imaging E-Gel 48-Well 2% Agarose Gels
10.3. Open top cabinet and line Gel up in the viewfinder.
SECTION: Imaging E-Gel 48-Well 2% Agarose Gels
10.4. Turn off EpiWhite. Click AutoFocus, and then turn on Trans UV.
SECTION: Imaging E-Gel 48-Well 2% Agarose Gels
10.5. Once the image is focused and bright, click freeze and turn Trans UV off.
SECTION: Imaging E-Gel 48-Well 2% Agarose Gels
10.6. Print the image at 110% and save the image both as the original file and as a JPEG.
SECTION: Imaging E-Gel 48-Well 2% Agarose Gels
10.7. Clean the stage EGel was resting on with Windex and throw Gel away in non-RCRA waste.
SECTION: Making Ladder Dilution
7. Make the ladder dilution: 10uL water + 5uL 1Kb Plus DNA Ladder (green top)

For One Gel: (10uL x 4 wells) = 40 µL water + (5uL x 4 wells) = 20 µL ladder stock