Agarose gels

A. Safety issues

We are using Gel Red to stain our DNA. This is significantly less hazardous that ethidium bromide as you can read about at Biotium. However, it is still a good idea to assume that everything having to do with running gels is contaminated and should be treated as hazardous. This includes gel boxes, microwave, the computer and gel running equipment. Always wear gloves when working in the gel area, typing on the computer or handling gels. You can also wear a lab coat to protect your clothes.

Once you leave the gel area, remove your gloves so as not to contaminate the rest of the lab. It's a good idea to wash your hands.

If you use the gel documentation system to photograph the gel, you don't need to wear a face mask. However, you should always wear a mask when cutting out bands out from the gel.

B. Preparing the gel

% agarose: The higher % agarose the gel is, the slower the DNA runs. However, the bands are tighter, so it is sometimes easier to see small pieces at higher % agarose. We typically use 2% for DNA < 200 bp and 1% for DNA > 200 bp

Kinds of agarose: There are several kinds of agarose in the lab.

Seakem LE is the cheap stuff. It is useful for analytical gels for running PCR reactions to see if there is something there or not.
Seaplaque GTG is a low melting point gel to use when you want to get the DNA bands back out for cloning, sequencing etc.
Seakem Gold is used for large (>10 kb) DNA fragments.

Sizes of gel rigs: We have several sizes of rigs. There are casting trays for the small, medium and large rigs for pouring gels in the hood.

Small rigs can run to 12 samples/row and two rows. It takes 30 ml to make a shallow gel and 50 ml to make a deeper gel (to hold 50 ul using the 8 tooth comb).

The medium rigs can run up to 14 samples/row and 2 rows. It takes at least 60ml to make a thin gel.

The large rig can run up to 26 samples / row and 2 rows. This rig is nice for running half of a 96 well plate (2 x 24 plus size standards).

The extra large rig can run up 42 samples / row and 3 rows. We also have a pulsed field gel rig for large DNA fragments (>10 kb).

Pouring gels:

Set up the gel tray in gel box or pouring tray. Make sure the O rings are sealed and not rolled up between plastic tray and box. Put desired combs in holder locations. Include 1 lane for size standard in each row. Combs with more teeth make wells which hold less volume (you can calculate the volume from the well length x width x gel height).

Make up enough gel to fill tray in an erlenmeyer flask. You can make up a bigger batch of gel (300 ml) in 500 ml bottle and re-microwave as needed. For 1% gel add 1g of agarose for each 100 ml of running buffer (1x TAE). Microwave till agarose is dissolved and there are no bubbles. NOTE: Never microwave gel with gel red in it.

Pour required volume into erlenmeyer flask and cool slightly. Add gel red (in drawer under microwave: about 5 ul of 10,000x Gel red stock per 50 ml of gel gives enough staining essentially no background). Pour into gel tray and let cool.

Gels can also be stained after running. To post stain, add Gel red to running buffer to 0.5 ug/ml and stain the gel for a half hour.

C. Electrophoresis

DNA is negatively charged at the pH's we use. When an electric field is applied, DNA will move to the positive (red) pole. If the gel box is set up with the electric leads at the back, the DNA will run to the right and to the red lead.

Loading gel:

Set up gel box with electrical plugs towards the back. Turn solidified gel so the wells are on the left. Fill the gel box with the same buffer as used in making the gel (TAE) so about 1 mm of buffer covers the top surface of gel. Remove the combs.
Loading buffer is added to each sample prior to loading. The loading buffer is a sucrose or glycerol solution with dye in it. The sugar increases the density of the sample so it sinks into the well. The dye helps keep track of which lanes have been loaded. We typically use two kinds of loading dye: blue and orange. The blue dye is xylene cyanol and is made up in sucrose. (I make up size standard in this XYC). The blue XYC dye runs at about 500 bp in a 1% gel. It's location varies with gel concentration. Some people also include bromophenol blue which gives a 2nd blue/purple band at a larger size during the run. Orange G is orange and is made up in glycerol. (I typically use this for samples). Orange G runs at the front so you can prevent samples from running off the end. Orange G can be loaded from parafilm; XYC can not.

Loading from tubes: Add required loading dye to PCR tube (2 ul for 10-20 ul of PCR reaction or 5 ul for 50 ul PCR reaction). Vortex and spin down. Load desired volume into wells in gel. Load size standard.

Loading from parafilm: For 10-20 ul of sample, put 2 ul of orange G on parafilm in a row of orange dots. Take PCR tubes and film w/ Orange G to gel room. Remove desired volume of PCR reaction and mix with orange G on parafilm. Load into well. Repeat for each sample. For 50 ul PCR reactions, use 5 ul Orange G. Load size standard

Running the gel:

Put lid on gel box so that female plugs fit on male jacks. Black lead should be on left and red on right. Connect leads to power supply. Gels should not be run faster than 5 V / cm. If you take the distance between electrodes (in cm) and multiply by 5 this is max voltage. If run gels too fast, they will heat up and melt. Hotter running gels will resolve less well.

Turn on power supply (ON / OFF switch). Set voltage. Small gels run typically at 50-70 V. Larger gels can be run at 80-100 V. Check the current by changing the volt / amp switch to amp. There can be voltage but no current if there is break in circuit. Current is typically around 1/2 voltage if buffer concentration is correct.

Let gel run, checking periodically. Gels often start running slow but then speed up as things warm up. For a single row gel, run till orange G is about 3/4 to end. Turn off gel and take picture. Always take a picture of your gel, even if it is blank. Sometimes you can see things in photo which you can't see by eye. Always keep a record.