“DNA is the Flash but Proteins are the Cash of Biotech”
•pAmylase Restriction Digestion (Lab 8b)
~40 bp
Ellyn Daugherty
SMBCP, San Mateo CA
www.BiotechEd.com
www.SMBiotech.com
www.emcp.com/biotech
www.sargentwelch.com/biotech
The Amylase Project
Model of rDNA/protein Business
< Chapters 1-5 Basic SLOP
< Lab 5f Amylase PAGE (revised)
< Lab 6e Amylase Producing Bacteria
< Lab 6c Amylase Activity Assay
< Lab 6d Amylase ELISA and W Blot (new)
< Labs 7a/7b/7f/7g Amylase Spectrophotometry
< Lab 8b Restriction Digestion of pAmylase
< Lab 4j/8b/8g DNA Gel Electrophoresis (revised)
< Lab 8c pAmy Transformation of E. coli
< Lab 9c/9d Amy Ion-Ex Chromatography
< Lab 13i Amylase Gene PCR (new)
< Lab 8g/4h Genomic & plasmid DNA Isolation
We use lots of ASSAYS in Biotech
DNA
•
Presence – gel electrophoresis (RE Digests &
PCR), dot blot tests
•
Concentration – UV spectrophotometry, dot blot
tests
Protein
•
Presence – protein indicators,
spectrophotometry, PAGE, Western blot
•
Activity/Function - protein indicators, enzyme
activity assays, spectrophotometry
•
Concentration – protein indicators,
spectrophotometry, ELISA
•
Stability/Shelf-life or other characteristics
Genetic Engineering
uses
Recombinant Plasmids
For Amylase Project (Ch 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.
PCR and Restriction Digestion
each can confirm the
recombinant plasmid is “right.”
The pAmylase Plasmid
(Lab 8b Restriction Digestion)
pAmy is about 6700 bp long
and contains:
• The amylase gene
(from Geobacillus stearrnothermophilus)
>>> can PCR and run a gel
to recognize the gene
• Several restriction enzyme
recognition sites including:
• 1 for HindIII & 3 for BamHI,
>>> see evidence of this
using restriction digestion
and gel electrophoresis.
~40 bp
Agarose Gel Preparation
• Determine amount of agarose
(g) to use
• Mix it with 1X TAE (or other
electrophoresis buffer)
• Heat on medium power in
microwave until completely
dissolved
• Cool to 65°C
• Place well comb
• Pour agarose to about 7 mm
thick
• Allow 10-15 min to cool
• Add electrophoresis buffer
before removing comb
(Lab 4i)
The pAmylase Plasmid after RE Digestion
Draw in the expected
DNA fragments
produced during
restriction digestion by
HinDIII and Bam HI
Get even more help!
Ellyn Daugherty
SM Biotech Career Pathway
www.SMBiotech.com
www.BiotechEd.com
www.emcp.com/biotech
www.sargentwelch.com/biotech
Ellyn@BiotechEd.com
Amy Naum
VWR Ed/Sargent Welch
www.sargentwelch.com/biotech
Amy_Naum@vwreducation.com 585-321-9422
Holly Ahern
VWR Ed/Sargent Welch
www.sargentwelch.com/biotech
Holly_ahern@vwreducation.com 512-789-3155