Protocol/5

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Protocol 5: Restriction Digest
 
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Reagents:
 
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* Restriction enzyme buffer
 
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** Buffer information can be found at www.NEB.com using the NEBtools.
 
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* Restriction enzymes
 
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* MilliQ water
 
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* DNA to be digested
 
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Procedure:
 
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* Select a restriction enzyme buffer that is appropriate for BOTH of the enzymes you are using. See information sheets at front of lab for correct buffer and concentration.
 
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* The total volume of all enzymes in the reaction should be less than 10% of the final reaction volume. Enzymes usually are supplied at 10U/ul and 1ul will be more than enough for our digests.
 
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* Add components in the following order:
 
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**Water
 
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**Buffer
 
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**Bovine Serum Albumin-BSA (if needed)
 
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**DNA
 
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**Enzyme I
 
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**Enzyme II
 
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*An example of a typical 25 ul reaction would be:
 
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{|
 
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|MilliQ water || 15.5 ul
 
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|-
 
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|10x NEBuffer ||        2.5 ul
 
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|-
 
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|DNA (200 ng/ul) || 5.0 ul
 
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|-
 
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|XbaI      ||    1.0 ul 
 
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|-
 
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|PstI      ||    1.0 ul
 
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|}
 
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*Incubate at 37<sup>o</sup>C for one hour (longer is okay too).
 
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Notes:
 
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FastDigest (by Fermentas) enzymes use a single uniform buffer, and claim to work in 5 minutes.
 
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Latest revision as of 17:16, 26 October 2010