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