"
Team:TU Darmstadt/Notebook/Methods/Restriction digest
From 2014.igem.org
(Difference between revisions)
| (3 intermediate revisions not shown) | |||
| Line 4: | Line 4: | ||
<html> | <html> | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
<div id="leftNavi" class="grid_5"> | <div id="leftNavi" class="grid_5"> | ||
<nav> | <nav> | ||
| - | <ul class="menu"><li class="first"><a href="https://2014.igem.org/Team:TU_Darmstadt | + | <ul class="menu"><li class="first"><a href="https://2014.igem.org/Team:TU_Darmstadt" >Home</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Project" >Project</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results" >Results</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/PolicyandPractices" >Policy & Practices</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Achievements" >Achievements</a></li><li class="active"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook" >Notebook</a><ul class="menu2"><li class="first"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Labjournal" >Labjournal</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Materials" >Materials</a></li><li class="active last"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods" >Methods</a><ul class="menu3"><li class="first"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Agarose_gel_electrophoresis" >Agarose gel electrophoresis</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Dephosphorylation" >Dephosphorylation</a></li><li ><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/DNA_ligation" >DNA ligation</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/DNA_quantification_with_NanoDrop" >DNA quantification with NanoDrop</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/PCR_purification" >PCR purification</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/PCR_with_Pfu_polymerase" >PCR with Pfu polymerase</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Plasmid_preparation" >Plasmid preparation</a></li ><li class="active"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Restriction_digest" >Restriction digest</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/SDS-PAGE" >SDS-PAGE</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Bacterial_cell_culture" >Bacterial cell culture</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Cell_counting_plating" >Cell counting/plating</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Chemically_competent_cells" >Chemically competent cells</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Colony_PCR_with_Taq_polymerase" >Colony PCR with Taq polymerase</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Glycerine_stocks" >Glycerine stocks</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Heat_shock_transformation" >Heat shock transformation</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Protein expression" >Protein expression</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/hydrochloric_acid_extraction" >37% hydrochloric acid extraction</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Dichloromethane_extraction" >Dichloromethane extraction</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Methanol_extraction" >Methanol extraction</a></li><li class="last"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Ethanol_extraction" >Ethanol extraction</a></li></ul></li></ul></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Team" >Team</a></li><li class="last"><a href="https://2014.igem.org/Team:TU_Darmstadt/Gallery" >Gallery</a></li></ul> |
</nav> | </nav> | ||
</div> | </div> | ||
<div id="wikicontent" class="grid_19"> | <div id="wikicontent" class="grid_19"> | ||
| - | <!--TYPO3SEARCH_begin--><div id="c92" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">Restriction | + | <!--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: </b>Single DNA Digestion (20 µL batch) |
</p> | </p> | ||
<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
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.
