Determine the Identity of Unknown Plasmids Mission (Im)possible Determine the Identity of Unknown Plasmids

Lab Objectives Determine the identity of the plasmid in each labeled tube Use restriction enzymes and gel electrophoresis as tools to identify the unknown plasmids

Bacterial Plasmids A small piece of circular DNA found in many bacteria, separate from the main bacterial chromosomes

Bacterial Plasmids Pick up new plasmids from the environment Lose plasmids Help bacteria survive environmental stress Many plasmids contain resistant genes

Plasmids in Biotechnology Used as vectors (delivery systems) to insert foreign DNA into bacteria Bacteria will produce any gene related protein that is inserted into the plasmid

Plasmid Structure

Restriction Enzymes Enzymes that cut DNA at specific restriction sites (recognition sequences of DNA nucleotides) Found naturally in bacteria (evolved for protection) Over 3000 identified, 600 used commercially

Restriction Enzymes

Restriction Enzyme - EcoRI

Restriction Enzymes and Gene Cloning

Restriction Enzymes

Restriction Enzymes A “cut” is known as a digestion Cuts leave over-hanging single strands called “sticky ends”

Selection of a Restriction Enzyme

What two plasmids will we be working with? pSNAPf vector – 5,849 total base pairs 20 µg cost = $157 pMAL-5X vector – 5,677 total base pairs 20 µg cost = $228

pSNAPf vector (plasmid)

pMAL-5X (plasmid)

How will we use restriction enzymes to identify the unknown plasmids? X Z

Restriction Enzymes Name Bacteria Isolated From Recognition Site BamHI Bacillus amyloliquefaciens  EcoRI Escherichia coli  HindIII Haemophilus influenzae BstEII Bacillus stearothermophilus

Part I: Preparing the Restriction Digests and Controls Label the tubes with your initials! DNA Ladder X + enzyme X no enzyme Z + enzyme Z no enzyme

Part I: Preparing Restriction Digests and Controls

Part II: Electrophoresis of Restriction Digests and Controls Add 10 µL of dH2O to DNA Ladder (LAD) Add 8 uL of loading dye (LD) to each of the tubes: X +enz, X no enz, Z +enz, Z no enz Pool reagents by tapping the tubes on the lab bench Load 20 uL of each sample into a well of the gel and record which sample went into which well

Plasmids Configurations Most common Travels quickly for its size Most common Travels quickly for its size Most common Travels quickly for its size Does not move as quickly as supercoiled plasmids (also known as relaxed circular) Does not move as quickly as supercoiled plasmids Migrates very slowly Slowest