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Unformatted text preview: Lab 16 - DNA Technology II:
Restriction Endonucleases Rev. 2/21/2011 The Case (LM p. 31-32) Defendants (Parents and their young daughter)
• Acquired an infection believed to be nosocomial
• The infection led to complications and the need for an additional surgery
• The additional surgery was not covered by the parents’ insurance
• The parents were suing the hospital for the payment
• The parents state that many patients on the ward had a similar infection
• The case is an isolated incident
• Mask and gowning was a precautionary measure
• The parents should pay for the surgery in full
• Test the volunteer patients A, B, C, and D from the defendant’s ward
• If at least one of the volunteers has a similar infection, testing of all
patients will be mandated Introduction
Purpose: Isolate plasmids from bacteria
•Received 2 bacterial cultures containing plasmids. These bacteria were cultured
originally from patient A, B, C, and D.
•Isolated plasmid DNA from two of these cultures.
Purpose: Identify (map) the plasmid DNA molecules
•Cut plasmid DNA samples with EcoRI and PvuII restriction endonucleases.
•Run the digested DNA on an agarose gel.
•Determine the size of the plasmid fragments in base pairs.
•Compare the restriction patterns of the plasmids with that of the restriction
patterns of the plasmid from the defendant’s infection.
• Determine if any of the patients’ infections match the defendant’s. DNA II Lab Overview
First 50 minutes
1. Instructor set up Digestions of positive controls (before lab)
2. Students prepare 1% agarose gels in 1X TAE buffer
3. Students prepare Restriction digestions of their DNA samples
4. Take DNAII Quiz using clickers
Second/ Third Hour
1. Set up prepoured gels. Practice loading on practice gels. Load samples.
2. Run gel for 30 - 40 minutes (until BB dye is ~ 3 cm from the end)
3. Work on Data Analysis A during gel run and Review DNA Models (2D and 3D)
4. Instructors take pictures of gels. All students near the Gel Doc must wear
goggles. Each Team views gel under UV to see the DNA bands fluoresce green.
Save files as JPEG to desktop. Print 4 copies per page using “Picture Viewer.”
5. Load gel pictures to Bb sections for class. Complete Data Analysis B (see Bb)
outside of lab as a group.
6. Clean up and Check out Reagent Label
ID Defendant patient
DNA (+ control) Aliquot
1 tube in box on ice; DNA for
Instructor 2-log DNA ladder 2-log DNA 1 per section
Instructors load on gels 6X Loading Dye 6X Loading Dye or
6X LD 1 per Team (6)
For DNA digestions 1X Loading Dye 1X of 1X LD For practice gel pipetting
ONLY!!! EcoRI Master Mix EcoRI 2 tubes in box on ice
All Teams share PvuII Master Mix PvuII 2 tubes in box on ice
All Teams share SYBR safe SYBR 1 tube per section
Instructor pipettes Instructors – 10’ before Lab Starts
Remove lid from water bath and check that temperature is at 37o C
Set up Instructor control digestions as follows:
You have one tube of control DNA.
Label one pink tube I-PvuII and one white tube I-EcoRI. Prepare the PvuII
digestion first and allow it to start incubating before setting up the EcoRI digest.
Add 20 µl of control DNA to the PvuII tube. Add 20 µl of the 2X PvuII
Mastermix to the PvuII tube and incubate in the 37o C water bath.
Add 4 µl of control DNA to the EcoRI tube plus 16 µl of sterile TE. Add 20 µl of
the 2X EcoRI Mastermix to the EcoRI tube. Incubate tube in the 37o C water bath.
Incubate Instructor samples until student samples are ready.
After the incubation, add 8 µl of 6X LD to each tube. Instructors – Load samples on Team gels Load 10 μl of each sample onto each team’s gel (LM p. 54):
10 μl of PvuII digested control into well #8 on Teams 1, 3, and 5 gels
10 μl of EcoRI digested control into well #8 on Teams 2, 4, and 6 gels Load 10 μl of DNA standard (labeled 2-log DNA) into well #4 for each
group’s gel. This sample is already in dye. Lab Manual p. 54 Place samples in your rack in the order they are to be loaded.
Load your Gel: Right to Left with wells facing you
Instructor will load their samples first to demonstrate gel loading (gold)
8 Sample (Load 10 µl per well)
IE (patient ___) plasmid DNA + EcoRI
IU (patient ___) plasmid DNA uncut
IP (patient ___) plasmid DNA + PvuII
DNA standard (NEB 2-log DNA ladder)
2E (patient ___) plasmid DNA + EcoRI
2U (patient ___) plasmid DNA uncut
2P (patient ___) plasmid DNA + PvuII
Instructor Defendant DNA digested with EcoRI or PvuII Instructor add 4 μl of Sybr Safe to gel after
cooling and before pouring – Wear gloves!!! Students check flask with agarose after 5 minutes to see if they are cool to the touch.
Add comb to gel casting tray prior to pouring the gel.
If gels harden before pouring, remicrowave. Ensure that all the particles dissolve. Add
more SYBR safe. The % agarose determines how well
the DNA fragments are separated.
Higher % agarose gels are used to
separate out small DNA fragments,
and low % agarose gels are used to
separate out larger DNA fragments.
Migration of the
same fragment in
agarose. There is a limit to the lowest % of
agarose that you can use. What types of DNA ends do these two Restriction Enzymes produce? Do you understand how Restriction Enzymes (RE) cut DNA?
Do you understand the 5’ and 3’ nomenclature before and after RE digestion? What color do the DNA bands appear that are treated with SYBR Safe? DNA is mixed with loading dye before loading it onto an agarose gel (+SYBR Safe)
that is immersed in 1X TAE buffer 1. What is the Purpose of the Loading DYE?
2. What is the Purpose of Agarose?
3. What is the Purpose of Buffer?
4. What is the Purpose of Sybr Safe? A mutagen and toxic Figure from DNA Science What is the purpose of uncut DNA on a gel? Uncut plasmid DNA should be mostly in its supercoiled (sc) form.
You cannot compare the sizes of sc DNA with linear (fragments) of DNA. ...
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This note was uploaded on 05/22/2011 for the course BIO 205 taught by Professor O'neal during the Spring '08 term at SUNY Stony Brook.
- Spring '08