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Biotechnology Part II

Biotechnology Part II. The Plasmid Lab: Creating Recombinant DNA. Bacterial Plasmid. Circular piece of DNA Replicates independently Used as a VECTOR. Restriction Enzymes. Enzymes that cut DNA at specific nucleotide sequences.

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Biotechnology Part II

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  1. Biotechnology Part II The Plasmid Lab: Creating Recombinant DNA

  2. Bacterial Plasmid Circular piece of DNA Replicates independently Used as a VECTOR

  3. Restriction Enzymes Enzymes that cut DNA at specific nucleotide sequences. Different restriction enzymes cut at different nucleotide sequences.

  4. DNA Palindrome A perfect DNA palindrome!!!! NOT a DNA palindrome!!!! C CC G GG C A G A C G T C T G G GG C CC A DNA palindrome reads exactly opposite its complementary strand.

  5. Which of the following are DNA palindromes??? 1. GAATTC CTTAAG 3. AATGCC TTACGG 2. CGAAGC GCTTCG 4. AAATTT TTTAAA

  6. Let’s start your lab activity… Look at your enzyme sheet Notice how each restriction enzyme cuts at a different palindrome Color each restriction enzyme a different color Cut out the enzyme squares Ligase is a different enzyme… we’ll find out more about it tomorrow

  7. Lab-Plasmid DNA Number each of the columns 1-6 starting on the left. Write the number right next to the first base pair Cut out all six columns along the dotted line Tape the strips of DNA together length-wise. Place tape on the back of strips

  8. Lab- Find those palindromes! • Take the restriction enzyme cards we colored earlier- let’s start with Ava II • Search your plasmid DNA until you find the same sequence that appears on the card • Color the sequence on the paper plasmid the same color as the restriction enzyme. • Search the entire plasmid! The sequence may show up more than once! • Repeat for each enzyme! • Some sequences may overlap, so you can color over the other one • A sequence can start at the bottom of one strip and continue onto another

  9. Lab-Plasmid! Tape the plasmid together to form a circle. Your plasmid is now complete!!!

  10. Restriction Enzymes Restriction Enzymes cut with a staggered cut. Think of it like a zig-zag

  11. Sticky Ends Sticky Ends: Overhanging pieces of single stranded DNA If 2 pieces of DNA are cut with the same restriction enzyme, they will have the same sticky ends

  12. Lab- Cell DNA Take the sheet marked cell DNA and cut the strips along the dotted line Tape them together in numerical order This is not a plasmid, so we won’t tape it in a circle

  13. Lab-Cell DNA The gene we want to cut out is marked with horizontal lines. Your challenge Find a restriction enzyme that will cut out our gene of interest. We want an enzyme that will cut the cell DNA twice, but the plasmid DNA only once. WHY?????

  14. Put it all together… Since both were cut with the same restriction enzyme, they have the same sticky ends!! DNA Ligase: Enzyme that seals DNA fragments together Use tape as DNA ligase to attach your pieces of DNA. You have now created RECOMBINANT DNA!!

  15. Steps to Make Recombinant DNA Isolate the gene of interest Cut the gene out using a restriction enzyme Cut the plasmid using the same restriction enzyme Sticky ends are produced Use DNA ligase to attach sticky ends together

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