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

2. 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

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

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

5. 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

6. Plasmid Structure

7. 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

8. Restriction Enzymes

9. Restriction Enzyme - EcoRI

10. Restriction Enzymes and Gene Cloning

13. Restriction Enzymes

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

15. Selection of a Restriction Enzyme

16. 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

17. pSNAPf vector (plasmid)

18. pMAL-5X (plasmid)

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

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

25. 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

26. Part I: Preparing Restriction Digests and Controls

27. 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

28. 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