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

Info iconThis preview shows pages 1–3. Sign up to view the full content.

View Full Document Right Arrow Icon
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 ( ).
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
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
Background image of page 2
Image of page 3
This is the end of the preview. Sign up to access the rest of the document.

This document was uploaded on 01/03/2012.

Page1 / 5

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

This preview shows document pages 1 - 3. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online