DNA is the Show and proteins are the dough of biotech

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DNA is the Show and proteins are the dough of
biotech
Fri Aug 8/17 4-9 pm SMBTC
4 pm Welcome/Introductions/Plans and Logistics
430 pm Intro to DNA Studies Salmon Sperm DNA spooling (Lab 4b)
5 pm Collect saliva, Isolate DNA (Lab 13f)
6pm Set up Alu PCR (Lab 13g)
615 pm Working dinner
645 pm Isolate E. coli DNA (Lab 4h)
8 pm rDigestion pAmylase set up (Lab 8c)
9 pm Clean up

Sat 8/18 9 am 500 pm SMBTC
9 am Preview of the Day
915 am Prepare, load, and run gels (Lab 4 j)
1045 DNA Spectrophotometry (Lab 11i)
1130 Stain gels
Destain gels, photo analyze
Noon Photo and analyze the pAmy restriction digest gels
1 pm Lunch/discussion of gel results
2 pm More on DNA Concentration assays/spectrophotometry/gels
445 Concluding remarks
Ellyn Daugherty SM Biotech Career
Pathway www.SMBiotech.com www.BiotechEd.com www.em
cp.com/biotech AEEDaugher_at_aol.com www.sargentwelch
.com/biotech
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DNA Isolation/Analysis Important for RD

• RFLP - restriction enzyme analysis
• Gene Identification
• Recombinant DNA/genetic engineering
• DNA synthesis
• PCR
• DNA sequencing
• Microarrays
• All of these used in RD in pharmaceutical,
  industrial, agricultural, environmental
  biotechnology.
• A lot of these are also used in diagnostic
  biotechnology.

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DNA Structure is Important in DNA Isolation and
DNA Analysis

• Structure
• Large macromolecules (nucleotide necklaces)
• Sugar-Phosphate strands
• Diff sequence of nucleotides
• Functions
• Self replication
• mRNA production for protein production (gene
  expression)
• Structure allows
• Length and phosphate groups allow spooling.
• Sequence allows for unique recognition sites

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DNA Studies require DNA Isolation from Cells

• Get a sample of cells
• Explode them - use detergent, salt, /or heat
• Separate DNA from other contaminant molecules -
  use salts, enzymes, /or centrifugation
• Alcohol precipitation - usually with
  centrifugation. but
• Can spool long strands onto glass rods

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Lab 4b Salmon Sperm DNA Spooling
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Lab 4b Salmon Sperm DNA Spooling
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Lab 4b Salmon Sperm DNA Spooling
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Polymerase Chain Reaction (PCR)
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Polymerase Chain Reaction (PCR)
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Isolating DNA from Cheek Cells for PCR
Lab 13f
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Isolating DNA from Cheek Cells for PCR
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PCR for Alu Genotype Determination
Use either BABEC kit/protocol or Edvotek
kit/protocol Each has Master Mix and Primer Mix
and you provide cheek cell DNA and thermal cycler.
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Lab 13f
BABEC
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BABEC
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BABEC
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Isolating DNA from Bacteria Cells

• Get a colony of cells grow in broth culture
• Separate from broth with centrifugation
• Explode cells and separate DNA from other
  contaminant molecules - use detergent, salt,
  heat
• Alcohol precipitation - EtOH and spooling

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Isolating DNA from Bacteria CellsLab 4h
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Isolating DNA from Bacteria CellsLab
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Genetic Engineering uses Recombinant Plasmids
Restriction Digestion can confirm the recombinant
plasmid is right. For Amylase Project (Chapter
6-9) we trick E. coli cells to take up the
amylase gene (on a pAmy plasmid) and express it
to make large amounts of amylase.
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The pAmylase Plasmid
pAmy is 6700 bp long and has several restriction
enzyme recognition sites including 1 for
HindIII and 3 for BamHI gtgtgt it is easy to see
evidence of this using restriction digestion and
gel electrophoresis.
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The pAmylase Plasmid after RE Digestion
Draw in the expected DNA fragments produced
during restriction digestion by HinDIII and Bam HI
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Confirming the Presence of the pAmylase Plasmid
by RE DigestionLab 8c
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Confirming the Presence of the pAmylase Plasmid
by RE Digestion
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Running DNA Samples on Agarose Gels
Lab 4j
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Running DNA Samples on Agarose Gels
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Running DNA Samples on Agarose Gels
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How to Load the DNA Samples Gels
Load 22 uL of genomic DNA/loading dye
Load 23 uL of restriction digestions/ loading dye
Load 7 uL of Lambda DNA Standards/ loading dye
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Using the Spectrophotometer to Study Molecules
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How Molecular Concentration Affects Absorbance
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UV Spectrophotometry

• UV/VIS Specs have 2 light sources
• UV Deuterium 240-320 nm
• White Light Tungsten 350-700 nm
• UV light is good for
• Colorless molecules
• Small amounts of sample
• UV/VIS Spec Advantages
• Can retrieve unaltered sample
• Can use tiny amounts of sample
• Most include pre-programming
• Good for concentration and
• purity testing

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Determining DNA Purity and Concentration
DNA Concentration Equation
5 µg/mL is the concentration for transformation
DNA Purity Equation
The closer to 1.8 the better
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How to Use the UV Spectrophotometer

• Turn on and allow time for it to calibrate itself
  (keep lid closed)
• Select the program or wavelength desired
• Place a cuvette with blank solution (usually
  buffer) in holder
• Select blank and allow it to set 0.00 a.u.
• Replace blank with sample and select read
• Record absorbance of sample
• Repeat with other samples at the same wavelength
• Repeat blanking if changing the wavelength

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Other Uses of Spectrophotometry and
Spectrophotometers

• Spectrophotometry
• Technology is used in
• Protein Concentration Determinations
• ELISA Plate Readers
• Column Chromatography
• Genomics (gels capillary)
• FPLC
• HPLC
• Mass Spectrometry

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• Biotechnology Science for the New Millennium
• Chapter 1 What is Biotechnology?
• Chapter 2 The Raw Materials of Biotechnology
• Chapter 3 The Basic Skills of the Biotechnology
  Workplace
• Chapter 4 Introduction to Studying DNA
• Chapter 5 Introduction to Studying Proteins
• Chapter 6 Finding a Potential Biotechnology
  Product
• Chapter 7 Developing Assays for Biotech Products
• Chapter 8 Modeling the Production of a
  Recombinant Product
• Chapter 9 Bring a Biotechnology Product to
  Market

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Biotechnology Science for the New Millennium
Ellyn Daugherty aeedaugher_at_aol.com

• Text with Encore CD
• Lab Manual
• Instructors Guide for each
• Student Notebook available

www.BiotechEd.com for tour through the entire
curriculum, ancillaries, and websites