Team:TU Darmstadt/Notebook/Methods/Restriction digest

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<!--TYPO3SEARCH_begin--><div id="c92" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">Restriction digestion</h1></div><div><p><b>Short explanation:</b></p></div><div><p>In order to insert DNA fragments into plasmids via ligation it is necessary to digest both components with restriction enzymes.</p></div><div></div><div><p><b>Procedure:&nbsp;</b>Single DNA Digestion (20 µL batch)
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<!--TYPO3SEARCH_begin--><div id="c92" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">Restriction Digestion</h1></div><div><p><b>Short explanation:</b></p></div><div><p>In order to insert DNA fragments into plasmids via ligation it is necessary to digest both components with restriction enzymes.</p></div><div></div><div><p><b>Procedure:&nbsp;</b>Single DNA Digestion (20 µL batch)
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<p>The following is an example of a typical single restriction enzyme digestion.
<p>The following is an example of a typical single restriction enzyme digestion.

Latest revision as of 00:36, 18 October 2014

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Restriction Digestion

Short explanation:

In order to insert DNA fragments into plasmids via ligation it is necessary to digest both components with restriction enzymes.

Procedure: Single DNA Digestion (20 µL batch)

The following is an example of a typical single restriction enzyme digestion.

Mix:

- 14 µL nuclease-free water

- 2 µL 10x NEBuffer

- 1 µL Restriction Enzyme (10 u)

- 1 µL DNA Sample (1µg) 

Incubate for 30 min at 37°C.

Inactivate enzymes by incubating at 80°C for 20 min.

Use 5 µl of the sample and add 1 µl 6X Loading Dye to proceed a agarose gel electrophoresis.

Larger scale restriction enzyme digestions can be accomplished by scaling this basic reaction proportionately.

Procedure: Multiple Restriction Enzyme Digestion (20 µL batch)

Use the optimal buffer supplied with one enzyme if the activity of the second enzyme is acceptable in that same buffer. NEB Buffer 4 usually works well for every used enzyme. Follow the single restriction enzyme digestion by using 1 µL of the additional enzyme and take 1 µL less nuclease-free water.