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“DNA is the Flash but Proteins are the Cash of Biotech ”

pAmylase Restriction Digestion (Lab 8b). “DNA is the Flash but Proteins are the Cash of Biotech ”. ~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.

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“DNA is the Flash but Proteins are the Cash of Biotech ”

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  1. pAmylase Restriction Digestion (Lab 8b) “DNA is theFlashbut Proteins are theCashof Biotech” ~40 bp Ellyn Daugherty SMBCP, San Mateo CA www.BiotechEd.com www.SMBiotech.com www.emcp.com/biotech www.sargentwelch.com/biotech

  2. 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

  3. 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

  4. 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.”

  5. 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

  6. Agarose Gel Preparation (Lab 4i) • 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

  7. The pAmylase Plasmid after RE Digestion Draw in the expected DNA fragments produced during restriction digestion by HinDIII and Bam HI

  8. 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

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