Download Electrophoresis of DNA

Name______________________________
Date_________Per_____
Agarose Gel Electrophoresis DNA –Restriction Enzyme Cutting
Lab Group Team Members ______________________________
______________________________
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In the previous lab you worked with samples of DNA that had already been cut by restriction enzymes. In this
lab, you will use restriction enzymes to cut DNA samples yourself. You must complete the Electrophoresis of
DNA lab before beginning this one.
Restriction enzymes are enzymes (proteins that act as molecular tools) which cut DNA. Each restriction enzyme
cuts DNA at a particular nucleotide sequence, acting like molecular scissors. For instance, one enzyme that you
will use in this lab, EcoR I, cuts DNA at the sequence 5’-…GAATTC…-3’. This sequence of DNA is called the
recognition site for EcoR I.
Whenever EcoR I encounters this sequence in a strand of DNA, it cuts, breaking the sugar phosphate backbone
between the G and A on both strands of the DNA. Each enzyme cuts DNA in a predictable and reproducible
manner. Since the early 1970’s, hundreds of restriction enzymes have been discovered and catalogued according
to their recognition sites. Thus it is possible to choose from a library of these enzymes to cut DNA at chosen
sequences.
Restriction enzymes are produced by and derived from various bacteria. Their funny names come about from the
following rules:
EcoR I -------------An enzyme derived from Escheria coli (E. coli)
----------------------The particular enzyme among several produced by this strain
----------------------------------A particular strain of this bacteria (strain RY13)
---------------------------------------------First two letters of the species name of the bacterium
---------------------------------------------------------First letter of the genus name of the bacterium
Bacteria have restriction enzymes so that they can disable “alien” DNA of bacteriophages (viruses which infect
bacteria). The bacteria give their own DNA a cloak of protection by modifying the recognition sequence DNA in
their own genome. Then the restriction enzyme in the cell cuts the unprotected DNA of the invader.
Note: One unit (U) of activity is usually defined as the amount of enzyme required to digest 1g of lambda DNA
to completion in one hour, in the preferred enzyme buffer at the optimal temperature for that enzyme (usually 37).
After DNA has been cut or digested with restriction enzymes, what you get is DNA pieces of varying sizes. These
can be separated in an agarose gel by electrophoresis. One important part of characterizing the pieces is to
determine their sizes. The simplest “ruler” for measuring DNA pieces in gels is to compare them to pieces of
known size run in the same gel.
Since the sizes of pieces resulting from digesting of phage lambda DNA were some of the first ever determined,
these became the ruler of standard for determining the sizes of unknown pieces in pretty much all later research,
and are still used in research labs today. The Hind III digest (marker II) of lambda is one of the most popular for
use as a size standard. Estimation by eye works pretty well, but the sizes can be determined with greater accuracy
by using a semi-log plot.
Restriction Enzyme Cutting of DNA
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Safety Hazards:
No Food or Drink
Electrical
Biohazard
Chemical
Glassware
Materials:
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Gloves
Goggles
Electrophoresis Gel Box
2% Agarose Gel with the wells at the END
Buffer
Power Supply
P-20 Micropipette
P-10 Micropipette
Medium Micropipette Tips
Small Micropipette Tips
Waste Container
Microtube Rack
Sharpie
Microtubes
Freezer Block Tube Rack
Sterile Distilled H20
10x buffer restriction enzyme (React)
Uncut DNA
Enzyme BamH I
Enzyme EcoR I
Enzyme Hind III
Sample Loading Dye/Buffer
EcoR I + Hind III-cut DNA
Heat Block or waterbath set to 37
Staining Tray (labeled with your group names or number)
Fast Blast DNA Stain
1 Acetate sheet per person
Restriction Enzyme Cutting of DNA
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Procedure:SamplePreparation
1) Wash your table with disinfectant cleaner.
2) Put on gloves and goggles.
3) Label FIVE empty microtubes 1, 2, 3, 4, and C.
4) Using a P-10 micropipette, follow the Sample Preparation Chart to micropipette all the reagents in to the
proper tubes. START with sterile distilled H20. Keep all reagents on ice.
Micropipette SLOWLY, some of the reagents are very viscous (thick). In order to get all of each reagent out of
the micropipette tip, place the tip into the bead of sterile distilled H20 and plunge in and out a few times to
“wash” all the substance out of the tip. Change tips after each sample. WHEN IN DOUBT, CHANGE TIPS!
Restriction Digest Sample Preparation Guide: Check off each substance on the chart as you go.
Tube 1
Tube 2
Tube 3
Tube 4
Add to Tube: (day one)
Sterile Distilled H20
10.5L
8.5L
8.5L
8.5L
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10X Buffer Restriction Enzyme
1.5 L
1.5 L
(React 2) buffer
10X Buffer Restriction Enzyme
1.5 L
1.5 L
(React 3) buffer
Uncut DNA
3L
4L
4L
4L
Enzyme BamH I
1 L
Enzyme EcoR I
1 L
Enzyme Hind III
1 L
TOTAL VOLUME
15 L
15 L
15 L
15 L
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 Mix tube contents gently.
 Centrifuge for 3 seconds.
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 Incubate at 37 C for at least 30
minutes or overnight.
 The next day, add Sample
Loading Buffer.
 Centrifuge for 3 seconds.
Sample Loading Dye/Buffer(day
3L
3L
3L
3L
two)
TOTAL VOLUME
18 L
18 L
18 L
18 L
Known Sample Preparation Guide:
Add to Tube: (day one)
STE Salt Buffer
(III) EcoR I + Hind III-cut DNA
 Vortex tube contents gently.
 Centrifuge for 3 seconds.
 Store in the freezer overnight.
 The next day, add Sample Loading
Buffer.
 Centrifuge for 3 seconds.
Sample Loading Dye/Buffer(day two)
TOTAL VOLUME
Tube C
10L
4L
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3L
17 L
Restriction Enzyme Cutting of DNA
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Procedure:Electrophoresis
1) Place the power supply on your lab table, but within reach of an electrical outlet.
2) Plug the power supply in, but make sure it is turned off.
3) Place the gel box where you can easily reach it to load the samples, but also within reach of the power supply.
(NOT on the edge of your lab table, but NOT in the middle either. Once you start loading the samples you
cannot move the gel box).DO NOT PLUG IT IN YET
4) Place the gel deck/agarose gel into the gel box, place the wells toward the negative end of the gel box.
5) Pour buffer into the gel box up to the fill line. The buffer should cover the gel by about 1-2 mm.
6) Keeping your samples in order, load 15L of each sample 1,
2, 3, 4, C, into separate wells in the gel.
7) Record on your lab sheet where each sample is placed into the gel (“before” diagram on p.5)
8) Place the lid on the gel box.
9) Plug the gel box into the power supply.
10) Turn on the power supply to 100V. (for at least 45 minutes) If you do not see bubbles, check all the wire connections, and the outlet.
11) Shut off the power supply.
12) Unplug the gel box from the power supply.
13) Carefully remove the lid of the gel box by lifting straight up.
14) Wearing gloves: carefully lift the gel deck out of the gel box and slide the gel into a staining tray.
15) Pour Fast Blast over the gel so that it covers the entire gel.
16) Place your staining tray on the rocker platform in the back room and leave it there OVERNIGHT.
17) Carefully pour the fast blast back into the original container.
18) Slide your gel into a ziploc bag.
19) Use the light box in the back room to identify all the bands of DNA. (There are some VERY FAINT bands,
so look carefully.
20) EACH PERSON: Place an acetate sheet on top of the gel and use a sharpie to record results. (tape to p.5)
21) Rinse and dry the gel box and gel deck Put away all equipment.
22) To dispose of the gel, place it into the garbage. Disinfect your table with Rubbing Alcohol, then wash your
hands well.
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Observations
Before Electrophoresis
After Electrophoresis
Tape Acetate Sheet Here
Negative End - - - - - - - - - - - -
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Positive End + + + + + + + + + +
Data Table
Sample
Observation: distance each band traveled in mm
1
2
3
4
C
Restriction Enzyme Cutting of DNA
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Questions
1. What is a restriction enzyme?
2. How does it determine where to cut DNA?
3. About how many different restriction enzymes are there?
4. What is a bacteriophage?
5. One unit (U) of activity is usually defined as:
6. What are the two methods for determining the size of DNA bands?
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