Corum:digest

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m (New page: ==Overview== Standard polymerase chain reaction (PCR) protocol. ==Materials== For a 50 μL PCR reaction: * 35 μL H<sub>2</sub>O * 5 μL 10X PCR buffer * 5 μL 2mM dNTPs (each) * 1.5 μ...)
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*'''SC 14:47, 19 June 2012 (EDT)''':
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==Overview==
==Overview==
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Standard polymerase chain reaction (PCR) protocol.
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Standard DNA digest protocol (NEB restriction enzymes).
==Materials==
==Materials==
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For a 50 μL PCR reaction:
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For a DNA digestion of total volume V (in μL):
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* 35 μL H<sub>2</sub>O
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* ~0.8V DNA
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* 5 μL 10X PCR buffer
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* 0.1V 10X digestion buffer (1, 2, 3, or 4; NEB)
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* 5 μL 2mM dNTPs (each)
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* 0.1V 10mg/ml BSA
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* 1.5 μL 50mM MgCl<sub>2</sub>
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* 1 μL restriction enzyme 1
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* 1 μL 50μM sense primer
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* 1 μL restriction enzyme 2 (optional, for double digestion)
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* 1 μL 50μM antisense primer
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* 1 μL 5nM DNA template
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* 0.5 μL TAQ DNA polyermerase
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==Procedure==
==Procedure==
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# In a PCR tube, mix the components on ice in the order they are listed above.
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# Find appropriate enzymes(s), associated buffer, reaction temperature from [http://www.neb.com|NEB].
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# Perform the following thermocycling program:
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# 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.
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## 95 °C 5 min
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# Calculate the volume of 10mg/ml BSA and 10X digestion buffer, which are both 0.1V.
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## 95 °C 30 s
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# Add the reaction components together in order listed above. Mix gently and spin.
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## T<sub>H</sub> 30 s
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# Incubate at reaction temperature for 3 hr to overnight.
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## 72 °C 1 min for each 1 kb PCR product
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## Repeat steps 2-4 a total of 12-36 times (24 is standard).
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## 72 °C 5 min
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## 12 °C hold
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==Notes==
==Notes==
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==Contact==
==Contact==
* [http://openwetware.org/wiki/User:Sean_P_Corum Sean P Corum]
* [http://openwetware.org/wiki/User:Sean_P_Corum Sean P Corum]
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* '''SC 17:44, 18 June 2012 (EDT)''':
 
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
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[[Category:In vitro]]
[[Category:In vitro]]
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[[Category:PCR]]
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[[Category:Digestions]]

Revision as of 14:47, 19 June 2012

  • SC 14:47, 19 June 2012 (EDT):

Contents

Overview

Standard DNA digest protocol (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 [1].
  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 and books

  1. Sambrook, J and Russell, DW (2001) Molecular Cloning: A Laboratory Manual (Volume II) - Cold Spring Harbor Laboratory Press ISBN 0879695773

Contact

or instead, discuss this protocol.

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