Corum:digest

(Difference between revisions)
 Revision as of 13:47, 19 June 2012 (view source)m ← Previous diff Revision as of 13:49, 19 June 2012 (view source)m Next diff → Line 2: Line 2: ==Overview== ==Overview== - Standard DNA digest protocol (NEB restriction enzymes). + Standard DNA digest protocol using [http://www.neb.com | NEB] restriction enzymes. ==Materials== ==Materials== Line 15: Line 15: ==Procedure== ==Procedure== - # Find appropriate enzymes(s), associated buffer, reaction temperature from [http://www.neb.com|NEB]. + # Find appropriate enzymes(s), associated buffer, reaction temperature from [http://www.neb.com | NEB]. # 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. # 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. # Calculate the volume of 10mg/ml BSA and 10X digestion buffer, which are both 0.1V. # Calculate the volume of 10mg/ml BSA and 10X digestion buffer, which are both 0.1V. Line 33: Line 33: - #{{FormatRef|Sambrook, J and Russell, DW|2001|Molecular Cloning: A Laboratory Manual (Volume II)| | |Cold Spring Harbor Laboratory Press}} ISBN 0879695773 + #[http://www.neb.com | New England Biolabs] ==Contact== ==Contact==

Revision as of 13:49, 19 June 2012

• SC 14:47, 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.