1 / 10

Understanding Restriction Digestion in Biotechnology: Using EcoRI and SmaI Enzymes

This guide explores the fundamentals of restriction digestions in biotechnology, specifically utilizing restriction enzymes such as EcoRI and SmaI to cut DNA. The concept of sticky and blunt ends is explained, highlighting EcoRI's recognition site (5'-GAATTC-3') and PstI (5'-CTGCAG-3'). The workflow for digesting a plasmid vector (2.9 kb) with an insert (1.1 kb) is detailed, along with necessary components like DNA, enzymes, and buffers. A thorough understanding of PCR methodology, including primer design for red fluorescent protein, is also provided.

emmanuel-frank
Download Presentation

Understanding Restriction Digestion in Biotechnology: Using EcoRI and SmaI Enzymes

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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)

More Related