Biotechnology Part II

Biotechnology Part IIThe Plasmid Lab: Creating Recombinant DNABacterial PlasmidCircular piece of DNAReplicates independentlyUsed as a VECTOR

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

DNA PalindromeA DNA palindrome reads exactly opposite its complementary strand.C A G A CC C C G G GNOT a DNA palindrome!!!!A perfect DNA palindrome!!!!G T C T GG G G C C CWhich of the following are DNA palindromes???1. GAATTC CTTAAG

3. AATGCC TTACGG

2. CGAAGC GCTTCG

4. AAATTT TTTAAALets start your lab activityLook at your enzyme sheetNotice how each restriction enzyme cuts at a different palindromeColor each restriction enzyme a different colorCut out the enzyme squaresLigase is a different enzyme well find out more about it tomorrowLab-Plasmid DNANumber each of the columns 1-6 starting on the left.Write the number right next to the first base pairCut out all six columns along the dotted lineTape the strips of DNA together length-wise. Place tape on the back of strips123456Lab- Find those palindromes!Take the restriction enzyme cards we colored earlier- lets start with Ava IISearch your plasmid DNA until you find the same sequence that appears on the cardColor 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 oneA sequence can start at the bottom of one strip and continue onto another

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

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

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

Lab- Cell DNATake the sheet marked cell DNA and cut the strips along the dotted lineTape them together in numerical orderThis is not a plasmid, so we wont tape it in a circleLab-Cell DNAThe 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?????Put it all togetherSince 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!!

Steps to Make Recombinant DNAIsolate the gene of interestCut the gene out using a restriction enzymeCut the plasmid using the same restriction enzymeSticky ends are producedUse DNA ligase to attach sticky ends together