1. Restriction Enzymes and Gel Electrophoresis

2. What is a Palindrome? A palindrome is anything that reads the same forwards and backwards: A palindrome is anything that reads the same forwards and backwards: English palindromes: English palindromes: mom mom dad dad Rats live on no evil star. Rats live on no evil star.

3. DNA Palindromes Palindromes are defined as any double stranded DNA which when read 5’ to 3’ and 3’ to 5’, they read the same. Palindromes are defined as any double stranded DNA which when read 5’ to 3’ and 3’ to 5’, they read the same. The EcoRI cutting site: 5'-GAATTC-3' 3'-CTTAAG-5' The HindIII cutting site: 5'-AAGCTT-3' 3'-TTCGAA-5'

4. Restriction Enzyme Review DNA scissors or cuts DNADNA scissors or cuts DNA Two types of cuts:Two types of cuts: Blunt endsBlunt ends Sticky endsSticky ends Each enzyme has a specific palindromic sequence that it recognizesEach enzyme has a specific palindromic sequence that it recognizes

5. How many Fragments? DNA: AGTCGTAGCTCGATC CC GGGCTGATCGAA TCAGCATCGAGCTAG GG CCCGACTAGCTT Restriction Enzyme: Sma I cuts at …C C C G G G… …G GG C C C… How many fragments? AGTCGTAGCTCGATCCC GGG CTGATCGAA TCAGCATCGAGCTAGGG CCC GACTAGCTT Restriction site Cut site

6. Step 2: Gel Electrophoresis Separates DNA (or RNA or Protein) fragments based on size and chargeSeparates DNA (or RNA or Protein) fragments based on size and charge Each sample goes into its own well and then has its own laneEach sample goes into its own well and then has its own lane Larger fragments move slower through the gelLarger fragments move slower through the gel

7. Electrophoresis = movement of charged particles using an electric fieldElectrophoresis = movement of charged particles using an electric field DNA is negatively charged so it moves towards the positive end of the boxDNA is negatively charged so it moves towards the positive end of the box Step 2: Gel Electrophoresis

8. Length of Fragments Number of base pairsNumber of base pairs 1 Base Pair = 2 Nucleotides1 Base Pair = 2 Nucleotides Examples: How many base pairs?Examples: How many base pairs? DNA Ladder = sample made up of DNA with known lengths Blunt ends: ATGTC TACAG Sticky ends: AGCTTGTA ACATTCGA ATAT

9. Gel Electrophoresis Wells

10. Gel Electrophoresis Wells

11. Gel Electrophoresis + - Direction of DNA Travel Wells Small Large DNA Ladder

12. G CTTAA AATTC G 1 Digestion 2 Annealing of sticky ends 3 Ligation Ligase G CTTAA AATTC G EcoRI R. E.s and DNA Ligase Can be used to make recombinant DNA GAATTC CTTAAG GAATTC CTTAAG G CTTAA AATTC G 4 Recombinant DNA