# Corum:digest

(Difference between revisions)
 Revision as of 14:31, 19 June 2012 (view source)m (New page: ==Overview== Standard polymerase chain reaction (PCR) protocol. ==Materials== For a 50 μL PCR reaction: * 35 μL H2O * 5 μL 10X PCR buffer * 5 μL 2mM dNTPs (each) * 1.5 μ...)← Previous diff Current revision (14:53, 19 June 2012) (view source)m (3 intermediate revisions not shown.) Line 1: Line 1: + '''SC 14:53, 19 June 2012 (EDT)''': + ==Overview== ==Overview== - Standard polymerase chain reaction (PCR) protocol. + Standard DNA digest protocol using [http://www.neb.com NEB] restriction enzymes. ==Materials== ==Materials== - For a 50 μL PCR reaction: + For a DNA digestion of total volume V (in μL): - * 35 μL H2O + * ~0.8V DNA - * 5 μL 10X PCR buffer + * 0.1V 10X digestion buffer (1, 2, 3, or 4; [http://www.neb.com NEB]) - * 5 μL 2mM dNTPs (each) + * 0.1V 10mg/ml BSA - * 1.5 μL 50mM MgCl2 + * 1 μL restriction enzyme 1 - * 1 μL 50μM sense primer + * 1 μL restriction enzyme 2 (optional, for double digestion) - * 1 μL 50μM antisense primer + - * 1 μL 5nM DNA template + - * 0.5 μL TAQ DNA polyermerase + ==Procedure== ==Procedure== - # In a PCR tube, mix the components on ice in the order they are listed above. + # Find appropriate enzymes(s), associated buffer, reaction temperature from [http://www.neb.com NEB]. - # Perform the following thermocycling program: + # 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. - ## 95 °C 5 min + # Calculate the volume of 10mg/ml BSA and 10X digestion buffer, which are both 0.1V. - ## 95 °C 30 s + # Add the reaction components together in order listed above. Mix gently and spin. - ## TH 30 s + # Incubate at reaction temperature for 3 hr to overnight. - ## 72 °C 1 min for each 1 kb PCR product + - ## Repeat steps 2-4 a total of 12-36 times (24 is standard). + - ## 72 °C 5 min + - ## 12 °C hold + ==Notes== ==Notes== Line 35: Line 30: ==References== ==References== - '''Relevant papers and books''' + '''Relevant papers, books, and websites''' - #{{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== * [http://openwetware.org/wiki/User:Sean_P_Corum Sean P Corum] * [http://openwetware.org/wiki/User:Sean_P_Corum Sean P Corum] - * '''SC 17:44, 18 June 2012 (EDT)''': or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. Line 54: Line 48: [[Category:In vitro]] [[Category:In vitro]] - [[Category:PCR]] + [[Category:Digestion]] + + [[Category:Restriction Enzyme]]

## Current revision

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.