Double Digestion with NEBcloner

Find out how easy it is to get double digest recommendations with NEBcloner.

Script

In this video, you will learn how double digests are made even easier with a tool, NEBCloner.

NEBCloner provides a nice protocol taking into account buffer, temperature, additive, as well as star activity differences. Let's see how.

Open up NEBCloner and select digestion.

Next, choose the two enzymes that you would like to digest simultaneously.  Please note that the second enzyme is optional. The tool will give you a protocol with just one enzyme as well.

You can type the name of the enzyme or select it from the pull down menu.

Press show protocol. In the protocol, the tool chooses a common buffer because both enzymes have greater than 50% activity in a single buffer.

Please note down below there was space for additional notes which can be printed for your notebook.  Press print protocol.

Now, let's choose a scenario where a sequential digest is required. The protocol recommends that the first digestion be carried out with the enzyme buffer combination requiring the lowest amount of salt.

After the first digestion, the protocol provides you with information on how to adjust the salt concentration so that the second digest can be carried out without a DNA cleanup and buffer exchange step. In certain cases where a salt adjustment is not possible, a DNA cleanup step may be needed.

Things to note: NEBCloner tells you about the methylation sensitivities of each of the enzymes selected. In this case, BsmB1 may be affected by CPG methylation. It also links you to heat inactivation information.

And that's it. Simple but comprehensive. Use NEBCloner to help you with your double digests.

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