Restriction Digest

Today: Setup digest and incubate Before Thanksgiving: we will run

the gel electrophoresis and analyze the results

What are restriction enzymes?

Enzymes that are able to cleave at specific and unique DNA sequences

Produced by bacteria as a defense against bacteriophage and other foreign DNA

Named according to the bacteria that produce them

Restriction sites Restriction enzymes (RE) recognize specific

sequences called restriction sites (usually palindromic sequences e.g. TTCATGAA)

The longer the restriction site sequence the enzyme recognizes the less likely the sequence will occur more than once

Two types of ends are produced when a RE cuts DNA:

1. Blunt

2. Sticky

Enzymes used in our labEcoRI (from Escherichia coli strain R)

5’-NNNGAATTCNNN-3’ 5’-NNNG-3’ 5’-AATTCNNN-3’ 3’-NNNCTTAAGNNN-5’ 3’-NNNCTTAAG-5’ 3’-GNNN-5’ Sticky Ends

BamHI (from Bacillus amyloliquefaciens)

5’-NNNGGATCCNNN-3’ 5’-NNNG-3’ 5’-GATTCNNN-3’3’-NNNCCTAGGNNN-5’ 3’-NNNCCTAG-5’ 3’-GNNN-5’ Sticky Ends

HindIII (from Haemophilus influenzae)

5’-NNNAAGCTTNNN-3’ 5’-NNNA-3’ 5’-AGCTTNNN-3’3’-NNNTTCGAANNN-5’ 3’-NNNTTCGA-5’ 3’-ANNN-5’ Sticky Ends

http://www.phschool.com/science/biology_place/biocoach/red/cleave1.htmlhttp://highered.mheducation.com/olc/dl/120078/bio37.swf

What are plasmids?

Small, self-replicating (has origin of replication), extra-chromosomal, circular, double stranded DNA

3,000 – 8,000 bp long Often found in bacteria

plasmids carrying genes for antibiotic resistance

pAMP plasmid carries a gene for resistance to ampicilin

Uses of plasmids

Genetic Engineering, Biotechnology or more specifically Gene cloning

Our experiment Overall Purpose: to execute a restriction digest,

analyze the results from a gel electrophoresis, in order to construct a map of pAMP plasmid

Experimental design:

1. Setup the digestion of a plasmid using three restriction enzymes: EcoRI, BamHI and HindIII

2. Run samples on an agarose gel electrophoresis

3. Perform analysis of the results and construct a restriction map

Digestion of plasmid DNA with EcoRI, BamHI and HindIII

Tube # 1 DNA control - no enzyme added Tube # 2 DNA Digested with BamHI only Tube # 3 DNA Digested with BamHI + EcoRI Tube # 4 DNA Digested with BamHI + HindIII Tube # 5 DNA Digested with HindIII + EcoRI

Tubes were incubated at 37C degrees for one hour and stored at 4C degrees.

Gel Electrophoresis

Gel-like matrix (agarose or acrylamide) is used to separate molecules based on their size, with the use of an electric current

Acrylamide+Bisacrylamide gels are mostly used to separate proteins and nucleic acids

Agarose gels are used to separate nucleic acids

DNA is negatively charged so if electric current is applied DNA will migrate towards the positive pole

Thus, it is important to set up your electrodes correctly when running a gel.

Shorter DNA fragments migrate faster across the gel than longer DNA fragments which migrate slower

To determine the actual size of the fragments we use known standards (called marker or ladder)

Agarose Gel Electrophoresis: Making the Gel

Gel tray will be perpendicular, once the gel has solidified, place the gel tray parallel with the gel box, pour the running buffer in the gel box to cover the gel, then slowly remove the comb. Add you samples to the wells, making note of which well each sample is in.

add Gelstar DNA stain, then

Setting up the Apparatus• DNA is negatively

charged so if electric current is applied DNA will migrate towards the positive pole

• Thus, it is important to set up your electrodes correctly when running a gel

• Red is positive• Black is negative

Loading DNA into the Gel• Before we load the

samples on the gel we need to add tracking dye. The tracking dye is added to make the solution dense so that our sample will "sink" into the well and to make the samples visible during analysis

• Load samples into the wells using a micropipettor

Movement of DNA Fragments

• We will run the gel at 90V for about one hour, until the tracking dye migrates about 2/3 of the way through the gel

• At this stage the DNA band(s) cannot be seen, only the tracking dye is visible

Photographing the Results

• In order to see the bands of DNA we have stained the gel with a fluorescent dye which binds to DNA (Gelstar DNA stain) making the bands visible under UV light

• We will use the UVP illuminator to take a photograph of the gel

Analysis of the results and plasmid map construction

1. Determine the approximate sizes of the fragments generated in each tube

2. Construction of the restriction map of the plasmid

3000 bp2500 bp

500 bp

Plasmid X

3000 bp

Predicted restriction sites

http://www.wiley.com/legacy/college/boyer/0470003790/animations/agarose/agarose.htm

http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/virgel.html