Download Human Mitochondrial DNA

Restriction Enzyme Cleavage
of DNA
Carolina Kit
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
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
Background Information
• Use website
http://bioinformatics.dnalc.org/gmo/animation/gm
o.html
• Animations
How did these samples get cut?
Cutting and pasting A & B
Why can restriction enzymes be used for?
Transferring and storing A & B
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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
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
Naming of restriction enzymes
• Based on their origin and order of
discovery
• 1st E. coli EcoRI
Why Restriction
Enzymes are important:
Transforming Cells
Uptake and expression
of foreign DNA by a cell
Making
Recombinant DNA
Prep. For RE cleavage lab
1 week before
1. label tubes—8 of each
DNA
EcoRI
HindIII
2. Make more TAE buffer
Monday
1. Prepare 0.8% agarose solution (add __g
agarose to ___ml of TAE, heat until clear) and
then pour gels
2. Pool DNA—spin in microfuge
Preparing gels
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___ 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
Each Group will run 3 DNA
samples
• Vial lambda DNA
• Vial lambda DNA cut with EcoRI
• Vial Lambda DNA cut with HindIII
Gel loading—change in protocol
Make sure to record
what is in each lane in
your lab notebook
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