The Plasmid Lab: Creating Recombinant DNA

 Circular piece of DNA  Replicates independently  Used as a VECTOR

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

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

1. GAATTC CTTAAG 3. AATGCC TTACGG 2. CGAAGC GCTTCG 4. AAATTT TTTAAA

 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

 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

1. Take the restriction enzyme cards we colored earlier- let’s start with Ava II 2. Search your plasmid DNA until you find the same sequence that appears on the card 3. Color the sequence on the paper plasmid the same color as the restriction enzyme. 4. Search the entire plasmid! The sequence may show up more than once! 5. 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

 Tape the plasmid together to form a circle.  Your plasmid is now complete!!!

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

 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

 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

 The gene we want to cut out is marked with horizontal lines.  Your challenge  WHY?????  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?????

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

1. Isolate the gene of interest 2. Cut the gene out using a restriction enzyme 3. Cut the plasmid using the same restriction enzyme 4. Sticky ends are produced 5. Use DNA ligase to attach sticky ends together