1. RESTRICTION DIGESTION AND ANALYSIS OF LAMBDA DNA

2. SAFETY FIRST JUMP TO SLIDE 22 WEAR GLOVES
WASH HANDS WITH SOAP WHEN DONE
HAPPY BIRTHDAY
JUMP TO SLIDE 22

3. What was the point of this lab?

4. What is lambda(λ)? Why lambda?
Lambda is a bacteriophage, virus that infects bacteria
Inserts its nucleic acid into the host bacterial cell
Replicates rapidly inside host cells until the cells burst and release more phages
Harmless to man and other
eukaryotic organisms
SOOOO, excellent source of DNA for experimental study.

5. Lambda genome is about 48,000 bp
If linear lambda DNA is cut with HindIII, how many fragments will there be? Longest piece? Shortest piece?

6. What is a restriction enzyme?
Enzymes that cut DNA at specific places known as restriction sites
Also called endonucleases
Bacteria use them as a natural defense against bacteriophages
Biotechnology – cutting genes from one organism and pasting them into another – would not be possible without these enzymes

7. Enzyme Site Recognition
Restriction site
Palindrone
• Each enzyme digests (cuts) DNA at a specific sequence = restriction site
• Enzymes recognize 4- or 6- base pair, palindromic sequences
(eg GAATTC)
Fragment 2
Fragment 1

8. 5 vs 3 Prime Overhang • Generates 5 prime overhang
Enzyme cuts
• Generates 5 prime overhang
DNA from any organism cut with the same enzyme will produce complementary sticky ends
When mixed together, complementary bases will hydrogen bond
Ligase is needed to reform the phosphodiester bonds.

9. Common Restriction Enzymes
5’ GAATTC 3’
3’ CTTAAG 5’
EcoRI
Escherichia coli
5’ overhang
HindIII
Haemophilus influensae
PstI
Providencia stuartii
3’ overhang
5’ AAGCTT 3’
3’ TTCGAA 5’
5’ CTGCAG 3’
3’ CACGTC 5’

10. How is a restriction digest done?
Restriction Buffer provides optimal conditions for enzyme
Why incubate at 37°C?
Body temperature is optimal for these and most other enzymes
What happens if the temperature is too hot or cool?
Too hot = enzyme may be denatured
Too cool = enzyme activity lowered, requiring longer digestion time

11. How can we separate all those fragments of DNA?
Agarose gel electrophoresis
Agarose is purified agar
Derived from seaweed
agar provides a medium on which bacteria (and other microorganisms can grow)
agarose provides a “sieve” for separating DNA fragments by size
Large fragments travel slower than small fragments

12. “swimming through Jello”
Gel electrophoresis
DNA is negatively charged
when it’s in an electrical field it moves toward the positive side
DNA         – +
“swimming through Jello”

13. Agarose Electrophoresis Loading
• Electrical current carries negatively-charged DNA through gel towards positive (red) electrode
Buffer
Dyes
Agarose gel
Power Supply

14. How can we see the DNA fragments – DNA is not colored?
Stain them!
The stain we use is relatively nontoxic, easy to handle and dispose of
BUT it’s not very sensitive so in “real life” other types of stains are used
Loading dyes and tracking dyes do not stain DNA; they just help you see where your sample is and how far DNA fragments have probably traveled

15. How can we determine the sizes of the DNA fragments?
Run a standard in one of the wells; also called marker or ladder
Standard has been cut with restriction enzymes and the size in base pairs (bp) has been determined
Compare migration of fragments whose size is known to the migration of fragments whose size is unknown

16. How can we determine the sizes of the DNA fragments?
Create a standard curve using the migration of the marker DNA fragments
Determine the size of the unknown fragments from this graph
Semi-log graph paper will be needed

17. Analysis of Stained Gel
Determine
restriction fragment
sizes
Create standard curve using DNA marker
Measure distance traveled by restriction fragments
Determine size of DNA fragments
Identify the related samples

18. Molecular Weight Determination
Fingerprinting Standard Curve: Semi-log
Size (bp) Distance (mm)
23,000* 11.0 9, 6, 4, 2, 2,
*This fragment falls outside the linear portion of the curve. You may choose to exclude it from your best fit line

19. OVERVIEW Page 21 You can answer (3 questions)

20. LESSON 1
Page 24 Let’s answer the two questions in the middle of the page
Be sure to fill out the chart
Page 25 You can answer – 2 questions at the top and the 4 review questions at the bottom
Page 26 You can answer – 4 questions
Page 27 You can answer – 4 questions

21. LESSON 2 Page 30 You can answer – 5 questions
Page 32 At the top – Let’s answer that now

22. LESSON 3 follow procedure 2. a on page 35
Page 36 Do not attach your tracing here; hand in separately; if you have no data another group will share with you – be sure to document where the data came from
Page 37; if you followed directions the wells are in this order:
Lane 1 Marker (we know the size of these fragments; they
were cut with HindIII )
Lane 2 Uncut lambda
Lane 3 lambda cut with PstI
Lane 4 lambda cut with EcoRI
Lane 5 lambda cut with HindIII

23. LESSON 3 Measure from the front of the well to the front of the band
RECORD DATA ON PAGE 38
BE CAREFUL GEL IS FRAGILE!

24. LESSON 3 Page 39 You do – 6 questions Page 40 You do – 2 questions
Clarification step 3 use marker
Page 42 – Complete the graph – you may ignore the 23,000 bp piece when drawing the best fit line
Page 43 in the “estimated” columns also include the data from the first gel analysis in parenthesis; the number from the standard curve graph should not be in parenthesis
Page 44 You do – 4 questions

25. Lane 1: marker, lambda cut with HindIII
Lane 2: uncut lambda
Lane 3: lambda cut with PstI
Lane 4: lambda cut with EcoRI
Lane 5: lambda cut with HindIII

26. Lane 1: marker, lambda cut with HindIII; 7 sites, 8 pieces
Lane 2: uncut lambda; 48,502 bp
Lane 3: lambda cut with PstI; 28 sites, 29 fragments
Lane 4: lambda cut with EcoRI; 5 sites, 6 fragments
Lane 5: lambda cut with HindIII

27. WHY DIDN’T WE SEE ALL THE FRAGMENTS AS BANDS IN THE GEL?
SOME BANDS ARE SO CLOSE IN SIZE THEY DID NOT SEPARATE USING THIS PROTOCOL
SOME FRAGMENTS ARE SO SMALL THEY CAN NOT BE DETECTED
How could we get better results?
Change gel concentration
Longer run time
More sensitive DNA stain

28. EXPERIMENTAL ERROR
*Incorrect measurement
*Not using optimal temperature for enzyme for the right amount of time
*Some of sample did not enter well
*Stock solutions not kept on ice