Bi1x_2009_W4

Bi1x_2009_W4 - Bi 1x, Spring 2009 Week 3 Session 1 o...

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Page 1 of 5 Bi 1x, Spring 2009 Week 3 Session 1 o Restriction enzyme digestion Session 2 o Agarose gel electrophoresis Restriction enzyme digestion Restriction enzymes are enzymes that bind to specific DNA sequences and cleave (“digest”) the DNA at or next to the binding site. You will become familiar with restriction enzymes during this week’s lab sessions on both bulk and single molecule scales. Note: This document describes bulk restriction digests only. Materials: Sterile, ultrapure water pZE21-lacZ plasmid DNA 10X NEB2 Buffer 10X EcoRI Buffer 10X BSA Solution HindIII digested lambda phage DNA EcoRI, HindIII, and KpnI restriction enzymes will be supplied by your TAs when you are ready to use them. Background: Most useful restriction enzymes recognize 4-8 base pair restriction sites. These sites are symmetric, inverted repeats called palindromes. Shown below are the restriction sites of the three enzymes you will be using today: EcoRI, HindIII, and KpnI. Notice how the 5’ to 3’ sequence is identical on the top and bottom strands. EcoRI: HindIII: KpnI: Some enzymes, like KpnI, produce sequences with 3’ overhangs upon cleavage. Others, like EcoRI and HindIII, produce 5’ overhanging ends. Additional enzymes can produce blunt sequences. It is also important to remember that restriction sequences are not necessarily unique to an enzyme—multiple enzymes often have the same recognition sequence. To look up the recognition sequences of different enzymes, you can consult the New England Biolabs (NEB) REBASE database ( http://rebase.neb.com/rebase/rebase.html ).
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Page 2 of 5 Restriction enzymes are generally supplied as a given number of units. These units correspond to a metric of enzymatic activity, as specified by the manufacturer. Today, you will be using enzymes from NEB, which uses the following definition for a “unit”: One unit is defined as the amount of enzyme required to digest 1 ± g of ² DNA in 1 hour at 37°C in a total reaction volume of 50
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Bi1x_2009_W4 - Bi 1x, Spring 2009 Week 3 Session 1 o...

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