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| - | ==Restriction Enzyme Digestion==
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| - | A method that allows to create a restriction map of a given DNA fragment. Widely used to test for the correct integration of a cloned sequence into a vector.
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| - | ===Theory===
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| - | Restriction enzymes, also called restriction endonucleases, are enzymes naturally found in bacteria which serve as a protection against foreign DNA found in a cell which usually implies a phage infection. The enzymes introduce a double-strand cuts in the DNA leaving either blunt or sticky single-strand ends, recognizing specific, usually 4bp or 6bp palindromic sequences.
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| - | ===Practice===
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| - | * '''Master mix preparation'''
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| - | For 2.0μl of DNA solution add:
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| - | :* 0.5μl of each restriction enzyme (or the respective amount of water for the control with uncut plasmids)
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| - | :* 0.1μl of BSA - ''acetylated Bovine Serum Albumin enhances the performance of restriction enzymes''
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| - | :* 5.9μl of water
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| - | :* 1.0μl of 2×NEBuffer
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| - | * '''Procedure'''
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| - | :# Mix DNA solution with the suitable amount of the master mix. As a control, repeat the same procedure with uncut DNA (prepare a master mix that does not contain restriction enzymes).
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| - | :# Incubate at 37°C for 2 hours.
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| - | :# Perform [[Team:Cambridge/Protocols/Gel_Electrophoresis | gel electrophoresis]] (10μl of DNA-restriction enzyme mixture in each well). Remember about molecular weight markers that help to assess the size of separated DNA fragments.
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| - | :# Compare with predicted fragment sizes. You can create a restriction map using [http://biologylabs.utah.edu/jorgensen/wayned/ape/ ApE - A plasmid Editor].
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| - | *'''Tips on Restriction Enzyme Usage'''
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| - | :* Store restriction enzymes in the freezer, at around -20°C.
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| - | :* Never make up restriction digests with the restriction enzyme composing more than 1/10 of the final volume. The reason is that restriction enzymes are stored in glycerol, and at concentrations above 10%, glycerol not only inhibits the digestion but also changes enzyme specificity (star activity).
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| - | :* Check for the compatibility of the restriction enzymes chosen.
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| - | : To check if the two selected restriction enzymes can perform effective catalysis in the same solution, go to the website of [http://www.neb.com New England Biolabs] → select [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp? Double Digest Finder] → choose which restriction enzymes you want to use → follow the digest recommendations.
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| - | ===Safety===
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| - | The main safety considerations are related to gel electrophoresis, namely:
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| - | * The use of SYBR Safe dye, in staining the DNA on the agarose gel. As it is a relatively toxic substance, make sure that all gloves, tubes and pipette tips that were in any contact with the dye are treated as [https://2011.igem.org/Team:Cambridge/Safety#Waste_Disposal hazardous chemical waste].
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| - | * Heating the solution of agarose in TAE buffer in a microwave. Care must be taken to avoid superheating.
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| - | <center>'''[[Team:Cambridge/Protocols|Back to Protocols]]'''</center>
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