1. Restriction Enzyme Cleavage of DNA
Carolina Kit

2. Timeline Week Before: HW—DNA scissors, DNA goes to the races
Monday—pour gels
Tuesday—Lecture, Run gels with DNA, photograph gel
Monday—Lab write-up due

3. Write-up Annotate handout
Data draw a gel and mark each banding site, staple picture to lab that you turn in to me
Results and Discussion (do the graph!)—answer all part

4. Background Information
Use website
Animations
How did these samples get cut?
Cutting and pasting A & B
Why can restriction enzymes be used for?
Transferring and storing A & B

5. Important for this lab:
Endonucleases – enzymes that cut RNA or DNA at specific sites; restriction enzymes are endonucleases that cut DNA
Sticky cells – restriction fragments in which one end of the double stranded DNA is longer than the other; necessary for the formation of recombinant DNA
Restriction enzyme mapping – determining the order of restriction sites of enzymes in relation to each other
Restriction enzymes are used for transformation (we will do this soon):
Transformation – the uptake and expression of foreign DNA by a cell
Transduction – the use of viruses to transform or genetically engineer cells
Competent/competency – the ability of cells to take up DNA
Selection – the process of screening potential clones for the expression of a particular gene, for example, the expression of a resistance gene (such as resistance to ampicillin) in transformed cells
Transformation efficiency – a measure of how well cells are transformed to a new phenotype
Recovery period – the period following transformation where cells are given nutrients and allowed to repair their membranes and express the “selection gene(s)”
Beta-galactosidase gene – a gene that produces beta-galactosidase, an enzyme that converts the carbohydrate X-gal into a blue product
Green fluorescent protein – a protein found in certain species of jellyfish that glows green when excited by certain wavelengths of light (fluorescence)
Scale-up – the process of increasing the size or volume of the production of a particular product

6. Restriction Enzyme Info. (page 216-218)
rDNA (recombinant DNA)—the produced piece of DNA from inserted another piece of DNA
recognize specific sites to cut the DNA
Blunt ends—straight across
Sticky ends—one side of DNA is longer than the other, these overhangs allow for complementary matches between two DNA pieces cut by the same enzyme, the sticky ends match and pasting ma occur to produce an rDNA molecule
More than 1200 restriction enzymes discovered & isolated from bacteria
Read 5 3
Palindromic (example radar or GAATTC CTTAAG

7. Naming of restriction enzymes
Based on their origin and order of discovery
1st E. coli EcoRI

8. Why Restriction Enzymes are important: Transforming Cells
Uptake and expression of foreign DNA by a cell
Making Recombinant DNA

9. Prep. For RE cleavage lab
1 week before 1. label tubes—8 of each
DNA
EcoRI
HindIII
2. Make more TAE buffer
Monday
Prepare 0.8% agarose solution (add __g agarose to ___ml of TAE, heat until clear) and then pour gels
Pool DNA—spin in microfuge

10. Preparing gels ___ grams agarose Add up to ___mL buffer
Melt in microwave, let cool
Set up trays—use 6 well comb
Pour about 30-50mL into each tray
Add 1uL ethidium bromide

11. Each Group will run 3 DNA samples
Vial lambda DNA
Vial lambda DNA cut with EcoRI
Vial Lambda DNA cut with HindIII

12. Gel loading—change in protocol
Make sure to record
what is in each lane in
your lab notebook

13. Results and Discussion
Before you leave the lab make, sure you have your measurements for the table in 4c
Use the graph paper to graph the table in 4c as explained in 4d-4j
Attach the graph paper to your lab write-up