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Lab 8

Lab 8. Restriction Digestions. Biotechnology. Using a restriction enzyme to cut DNA. Restriction Enzymes. Sticky ends: EcoRI 5’-GAATTC-3’. G + AATTC. 3’-CTTAAG-5’. CTTAA-5’ G. PstI 5'-CTGCAG-3' CTGCA-3’ + G.

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Lab 8

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  1. Lab 8 Restriction Digestions

  2. Biotechnology Using a restriction enzyme to cut DNA

  3. Restriction Enzymes Sticky ends: EcoRI 5’-GAATTC-3’ G + AATTC 3’-CTTAAG-5’ CTTAA-5’ G PstI 5'-CTGCAG-3'CTGCA-3’ + G 3'-GACGTC-5‘ G 3‘-ACGTC Blunt ends: SmaI 5’-CCCGGG-3’ CCC + GGG 3’-GGGCCC-5’ GGG CCC

  4. Restriction Digestion of a plasmid Vector=2.9 kb Insert= 1.1 kb EcoRI Total= 4.0 kb After digestion with EcoRI you will get: 1 fragment 2.9 kb + 1 fragment 1.1 kb in a electrophoresis run

  5. Components of a restriction enzyme digestion reaction • DNA • Buffer • Enzyme • The volume of the reaction should be make up to the right volume with MilliQ (pure) water when necessary • Digestions are often performed in a 20 ul volume • Digestions are incubated at the optimum enzyme temperature (most of them, 37 C) • Manual: Pg. 86-90

  6. Plasmid Vector Green Red Fluorescent Protein gen

  7. Lab 9 • PCR

  8. The polymerase chain reaction (PCR) Direct Reverse Step 1: 30 sec 95oC Step 2: 30 sec 45-55oC Step 3: 1-2 min 72oC

  9. The polymerase chain reaction (PCR) Primers Direct primer for red fluorescent protein gene: 5’- CTC TAG AGG ATC CCC GGG TAC – 3’ Reverse primer for red fluorescent protein gene: 5’ – CGG CGC TCG AGT TGG AAT TCT AGA GTC GCG – 3’

  10. Components of the PCR Reaction • DNA • Primers • 4 Nucleotides • Taq Polimerase • Buffer • Steps of PCR Reaction • Initial denaturation: 1 Cycle • Denaturation • Annealing • Extension • 5. Final extension: 1 cycle • http://www.youtube.com/watch?v=eEcy9k_KsDI&feature=related • Manual: Pg. 90-97 1 cycle (25-30 times)

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