Corum:DNA Digestion

SC 14:53, 19 June 2012 (EDT):

Overview

Standard DNA digest protocol using NEB restriction enzymes.

Materials

For a DNA digestion of total volume V (in μL):

• ~0.8V DNA
• 0.1V 10X digestion buffer (1, 2, 3, or 4; NEB)
• 0.1V 10mg/ml BSA
• 1 μL restriction enzyme 1
• 1 μL restriction enzyme 2 (optional, for double digestion)

Procedure

1. Find appropriate enzymes(s), associated buffer, reaction temperature from NEB.
2. Calculate the total volume of the reaction by taking the DNA volume + 1(2) μL for a single (double) digestion reaction. Divide this number by 0.8 to get the total reaction volume, V.
3. Calculate the volume of 10mg/ml BSA and 10X digestion buffer, which are both 0.1V.
4. Add the reaction components together in order listed above. Mix gently and spin.
5. Incubate at reaction temperature for 3 hr to overnight.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

1. List troubleshooting tips here.
2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Relevant papers, books, and websites

1. New England Biolabs

Contact

• Sean P Corum

or instead, discuss this protocol.