Restriction Enzyme Cleavage of DNA

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Restriction Enzyme Cleavage of DNA

• Carolina Kit

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Timeline

• Week Before HWDNA scissors, DNA goes to the
  races
• Mondaypour gels
• TuesdayLecture, Run gels with DNA, photograph
  gel
• MondayLab write-up due

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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

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Background Information

• Use website http//bioinformatics.dnalc.org/gmo/an
  imation/gmo.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

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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 endsstraight across
• Sticky endsone 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 GAATTCCTTAAG

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Naming of restriction enzymes

• Based on their origin and order of discovery
• 1st E. coli? EcoRI

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Why Restriction Enzymes are importantTransformin
g Cells
Uptake and expression of foreign DNA by a cell
Making Recombinant DNA
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Prep. For RE cleavage lab

• 1 week before1. label tubes8 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 DNAspin in microfuge

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Preparing gels

• ___ grams agarose
• Add up to ___mL buffer
• Melt in microwave, let cool
• Set up traysuse 6 well comb
• Pour about 30-50mL into each tray
• Add 1uL ethidium bromide

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Each Group will run 3 DNA samples

• Vial lambda DNA
• Vial lambda DNA cut with EcoRI
• Vial Lambda DNA cut with HindIII

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Gel loadingchange in protocol

• Make sure to record
• what is in each lane in
• your lab notebook

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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