How to Prepare and Load a Standard DNA Ladder

In this video, you will learn how to prepare a standard DNA ladder and load it onto an agarose gel.

Script

In this video, I will demonstrate how to prepare and load a standard DNA ladder, one that does not come in a ready-to-load format.

 

1. Select Tubes or Plate

 

Because standard NEB DNA ladders are concentrated DNA solutions, they need to be diluted and a loading dye should be added prior to loading onto a gel.

 

To prepare your DNA ladder and your samples, you’ll need some small plastic tubes; 500 µl tubes work well. Because we often load a lot of samples at once, we use a 96 well plate.

 

You need about 1 µl of the prepared DNA ladder mix per millimeter of gel lane. Most standard gel lanes are around 5mm wide so 6 µl is a good volume to load and easy to prepare.

 

2. Prepare the Ladder

 

Now we can prepare our ladder. Add 4 µl of water to your tube or well, and then add 1 µl of the 6X purple loading dye without SDS, which is supplied with most of our DNA ladders. Mix by flicking the tube and spinning briefly.

 

Add 1 µl of the DNA ladder to the diluted loading dye mixture. Mix again by flicking the tube and spinning briefly to gather the prepared ladder at the bottom of the tube. The DNA ladder is then ready to load on your gel.

 

3. Load the Ladder

 

Slowly pipette the 6 µl of the prepared DNA ladder mix into the desired well. Load your prepared samples and then you are ready to run your gel in the appropriate migration conditions.

 

TIP: Scale Up Ladder Mix

 

The DNA ladder mix can be scaled up if you need to load multiple gel lanes. Simply prepare a 1X dilution of the 6X purple loading dye, no SDS, in advance by diluting it with water. For each gel lane that you’d like to run, add 5 µl of the 1X purple loading dye dilution into a microfuge tube. Then add 1µl of the DNA ladder. Flick the tube with your finger and spin it briefly again. The DNA ladder is then ready to load.

 

You want to avoid diluting the DNA ladder in water directly, without the loading dye present. When the ladder is in the presence of water without loading dye, some of the DNA fragments can denature. This is why it is important to add the ladder to the diluted loading dye mixture.

 

Alternatively, you can use TE buffer instead of water for any DNA ladder dilution to prevent DNA denaturation.

 

TIP: For Maximum Convenience

 

For your convenience, NEB also offers an array of DNA ladders available in ready to load formats. These ladders are really easy to use. Invert the vial a couple of times to mix, then just load directly onto your gel. That’s it.

 

Contact Our Technical Support

 

If you are ever unsure of how to use any of our products, you can always contact our technical support scientists by phone, or by emailing [email protected]

 

We’re always happy to help.

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