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Stage 1: Prepare your plasmids to be cut by restriction enzymes

Learn how to prepare plasmids for cutting by restriction enzymes, analyze the fragments through DNA electrophoresis, and engineer bacteria for insulin production. This process involves mixing plasmids with enzymes like BamH1 and HindIII to cut at specific locations, followed by loading fragments into a gel for separation based on size.

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Stage 1: Prepare your plasmids to be cut by restriction enzymes

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  1. Stage 1: Prepare your plasmids to be cut by restriction enzymes • Obtain the plasmids (pKAN and pAMP) P stands for plasmid pKAN = plasmid with antibiotic kanamycin resistance pAMP = plasmid with antibiotic ampicillin resistance • Mix plasmids with restriction enzymes • BamH1 and Hind III • Restriction enzymes cut the plasmids at precise locations

  2. Plasmid w/ kanamycin resistance (pKAN) Restriction enzyme: Hind III (cuts @ bp 234) pKAN = 4194 bp Restriction enzyme: Bam HI (cuts @ bp 2095) 1861 bp restriction fragment 2333 bp restriction fragment

  3. Thousands of plasmids in our samples

  4. K–(uncut plasmid)- means the restriction enzymes not added K+(digested plasmid)+ means the restriction enzymes were added How many plasmid fragments? How many plasmid fragments?

  5. Stage 2: Check to see if the restriction enzymes worked • DNA electrophoresis • Plasmid fragments are loaded into a gel • Connected to a power supply • Separates fragments based on their sizes • Smaller fragments travel further through the gel

  6. Electrophoresis: sizes DNA fragments

  7. Electrophoresis: sizes DNA fragments K+ K- A+ A- Known DNA markers 10,000bp 8,000bp 6,000bp 5,000bp 4,000bp 3,000bp 2,000bp Look at the lab handout and let’s predict the A+ fragments 1,500bp 1,000bp 500bp

  8. 2008 Data/Results

  9. We will then micropipette the plasmids

  10. Load the plasmids into an electrophoresis chamber

  11. Micropipet tip should be ABOVE the well NOT IN IT!!!!

  12. Micropipette tip punched right through the gel See dye under the wells

  13. NICE!

  14. Connect the electrophoresis to a power supply…DNA has a negative electric charge.

  15. So now what??? • Plasmids have been engineered for human uses. The human gene for insulin (red) can now be added to the plasmid. The bacteria will produce insulin for diabetics!

  16. So now what??? The bacteria with the recombinant DNA replicates, thus passing the insulin gene onto its offspring. Each cell now will produce insulin for humans to harvest and use.

  17. K+ K+ K+ K+ A+ A+ A+ A+ K- A- Marker DNA Marker DNA 10,000bp 8,000bp 6,000bp 5,000bp 4,000bp 3,000bp 2,000bp 1,500bp 1,000bp 500bp

  18. K+ K+ K+ A+ A+ A+ K+ A+ K- A- Marker DNA Marker DNA 10,000bp 8,000bp 6,000bp 5,000bp 4,000bp 3,000bp 2,000bp 1,500bp 1,000bp 500bp

  19. K+ K+ K+ K+ A+ A+ A+ A+ K- A- Marker DNA Marker DNA 10,000bp 8,000bp 6,000bp 5,000bp 4,000bp 3,000bp 2,000bp 1,500bp 1,000bp 500bp

  20. K+ K+ K+ A+ A+ A+ K+ A+ K- A- Marker DNA Marker DNA 10,000bp 8,000bp 6,000bp 5,000bp 4,000bp 3,000bp 2,000bp 1,500bp 1,000bp 500bp

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