Restriction Digest Laboratory

Slides:

Advertisements

Similar presentations

Stage 1: Prepare your plasmids to be cut by restriction enzymes Obtain the plasmids (pKAN and pAMP) P stands for plasmid pKAN = plasmid with antibiotic.

Advertisements

Outbreak Lab: In this lab, biotech procedures will be used to see if a sample of viral DNA is the deadly Alabama virus. The specific technique that you.

Restriction Digest Laboratory. Reminder You have transformed bacteria with plasmid DNA You have isolated plasmid DNA Today you will perform an RFLP analysis.

BIO-RAD Forensic DNA Fingerprinting Kit

Restriction Mapping.

DNA Analysis Using Agarose Gel Electrophoresis Day 1

Mini-Prep Plasmid Isolation and Identification. Page 3-53 in lab manual & handout.

Restriction Mapping of a Bacterial Plasmid (Danna and Nathans, 1971)

Restriction Mapping of Plasmid DNA. Restriction Maps Restriction enzymes can be used to construct maps of plasmid DNA Restriction enzymes can be used.

Making a Restriction Map

& Gel Plasmid Electrophoresis Mapping.

Gel Electrophoresis based on motion of charged molecules in an electric field toward the opposite charge. Agarose gels (for larger fragments of DNA) or.

(RFLP Electrophoresis)

Recombinant DNA Technology

Forensic DNA Fingerprinting: Using Restriction Enzymes.

POLYMERASE CHAIN REACTION AMPLIFYING DNA What do you need to replicate DNA? umZT5z5R8.

Restriction Analysis and Digestion of Lambda DNA.

DNA Fingerprinting Supplies from Iowa State Student Preparation DNA and Enzyme Preparation.

Feb 3, 2015 I can… Predict how DNA is used to solve real- world problems Reminders: February 4 th – All first semester work must be turned in, please check.

Background Genetic disorders are often the result of gene mutations. People with a mutant allele often have a family history of the disease. It is important.

Biology 22 Molecular Laboratory Report 1. Bacterial Transformation 2. Plasmid Isolation 3. RFLP Analysis RESULTS ONLY due on April 21 st, 2011 Discussion.

(RFLP Electrophoresis)

BIO-RAD Lambda DNA Kit, and BIO-RAD Forensic DNA Fingerprinting Kit Section Concepts: DNA Scissors Activity, restriction enzymes, gel electrophoresis,

Restriction Digestion and Gel Electrophoresis Laboratory.

DNA Science. Restriction Digest Restriction Digestion is the process of cutting DNA molecules into smaller pieces with special enzymes called Restriction.

Digesting DNA Using Restriction Enzymes

A Timed Presentation - do not click the mouse. Approximate Run Time - 2 minutes 38 seconds.

Biology 22 Molecular Laboratory Report 1. Bacterial Transformation 2. Plasmid Isolation 3. RFLP Analysis.

Recombinant DNA Bacterial Transformation Student Instructions Plasmid Digest.

Restriction Digest Laboratory Restriction fragment length polymorphism.

Restriction Mapping of Plasmid DNA. Restriction Maps Restriction enzymes can be used to construct maps of plasmid DNA Restriction enzymes can be used.

Recombinant DNA Techniques chapter 18 Part I techniques and their applications. 1. Restriction Digest (to be done in lab) 2.Southern Blot 3.Northern.

Objectives: Introduce the students to digest genomic DNA by restriction endonucleases. Observe the results of digestion on agarose gel electrophoresis.

LAB 6 DNA FINGERPRINTING. BUILD the GEL FRAME Position comb and lock in place in side slots.

A 2 nd year college student working in the lab for the summer forgot to label the Tube of plasmid DNA that he/she was given. So being the brilliant graduate.

1. isolated mRNA for protein A from rat liver 2. reverse transcribed mRNA to DNA 3. added “sticky ends” to create an insert 4. ligated the insert (rat.

CHPT. 20 DNA TECHNOLOGY & GENOMICS & AP LAB #6A. Restriction Enzymes - -found in bacteria, they prevent invasion by foreign bacteria -cut specific.

Restriction Digest Laboratory

Restriction Enzyme Digestion of Phage DNA

Objectives: 1- Introduce the students to digest genomic DNA by restriction endonucleases. 2- Observe the results of digestion on agarose gel electrophoresis.

Uses of Restriction Enzymes

Overview Wednesday Thursday Labs 12, 13 & 14 due March 7th

Lab 3: BUILDING THE pARA-R PLASMID

Laboratory 3: building a recombinant plasmid

Recombinant DNA Techniques chapter 19

Restriction Enzymes and Plasmid Mapping

DNA Technology Now it gets real…..

Confirmation of pARA–R Restriction Digest

DNA Fingerprinting DNA Restriction Student Instructions

Lab 8: PTC Polymerase Chain Reaction Lab

Recombinant DNA Techniques chapter 19

Biotechnology: Restriction Enzyme Analysis of DNA

Opening Activity: March 12, 2018

Mini-Prep Plasmid Isolation and Identification

Restriction Endonuclease

In search of a cancer gene

Southern Blotting DNA Fingerprinting

Southern Blotting DNA Fingerprinting

Southern Blotting DNA Fingerprinting

Restriction Digest Laboratory

Determine the Identity of Unknown Plasmids

Southern Blotting DNA Fingerprinting

Cloning a DNA segment from bacteriophage

Southern Blotting DNA Fingerprinting

Opening Activity: March 20, 2017

Bio-Rad Biotechnology Explorer™ DNA Fingerprinting Kit

Forensic DNA Fingerprinting Lab

Presentation transcript:

Restriction Digest Laboratory Restriction fragment length polymorphism

Reminder You have transformed bacteria with plasmid DNA You have isolated plasmid DNA Today you will perform an RFLP analysis & Confirm your Plasmid Isolation

This is the third and final section of your lab report. Digest plasmid DNA Determine number of cutting sites Determine location of cutting sites Determine size of fragments Present the “map” of the plasmid in your report The steps in BLUE you will complete outside of class as part of your data analysis.

What is: A restriction enzyme(s)? A restriction digest? An endonuclease We will focus on type II. A restriction digest?

Restriction Enzyme Digest

Examples of Restriction Enzymes Links to restriction enzymes: http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp http://www.accessexcellence.org/AE/AEC/CC/re_chart.php http://www.neb.com/nebecomm/EnzymeFinder.asp?

Gel Electrophoresis Following Digest

Analysis of Data Allows you to identify sizes of plasmid By comparing migration of digested plasmid To KNOWN SIZES of DNA.

Example of known sizes of DNA DNA Ladder or Markers

A map gives the size of fragments A map gives the number and position of cutting sites 60 800 600 JUST AN EXAMPLE Not your map! 1500 Plasmid map 1400

Remember Plasmid is Circular Circular DNA: the number of fragments=number (N) of cutting sites versus Linear DNA: number of fragments=N+1

Linear DNA Plasmid DNA 2 cutting sites 2 fragments 2 cutting sites 3 fragments

Today’s experiment Restriction of Digest of plasmid DNA using two restriction enzymes.

Please refer to page 10 of the handout (6 groups) Each Group set up a rack with: Reaction buffer water Plasmid DNA NotI for AM lab SfiI for AM lab Or AluI for PM lab HphI for PM lab Loading Dye Standard (marker or ladder) DNA Label four microfuge tubes 1→4 Must keep on ice

Pipette the samples as shown on page in handout—not lab manual.

After you are finished pipetting your samples Place samples at 37C for 1 hour After 1 hour you will be ready to load your gel

Restriction Digest AFTER 1 hour DIGESTION: You must add 5 ul 10X loading dye to your samples (not to the ladder (L)). Pre-heat all samples including ladder for 3-5 min. at 65C

Gel Electrophoresis Load 25 ul per well Run gel at 75 volts until the dye front is approximately half-way down gel. Take photograph