D. M. Dean and J. A. Wilder
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FILE S2
Laboratory Exercise Supplies for “Frankenplasmid Lab”
While the MATERIALS AND METHODS section gives an overview of student and staff
procedures, and FILE S1 describes the student protocol in detail, this file lists the
supplies that the staff must prepare to implement the module in their classroom.
Unless otherwise noted, chemicals were purchased from Sigma-Aldrich Corporation.
Abbreviations with quotes indicate how a tube is labeled in the laboratory manual
(FILE S1). Liquid volumes represent the minimum volume needed for a pair of students.
We strongly recommend giving students an excess of each solution in case an error is
made or they overpipet from their supply. We also keep extra hardware supplies on hand
(e.g. microcentrifuge tubes). See MATERIALS AND METHODS for further details about the
preparation of competent cells, plasmids, backup cultures, and PCR primers.
PRELABORATORY EXERCISE 1
Students are to complete this exercise before Week 1. They will need their
laboratory manual (FILE S1) and a computer with internet access.
WEEK 1, DAY 1
Each pair of students receives:
-A set of three Pipetman micropipettors (Gilson; P1000, P200, and P20) with
compatible tips
-Fine-tipped, permanent marker to label Eppendorf tubes and Petri plates
-Vortex Mixer (Fisher Scientific)
-2 LB-agar/kanamycin/tetracycline/X-gal plates (see “SOLUTIONS” below)
-Bunsen burner or ethanol lamp
-Lighter or matches
-Bacterial spreader and beaker filled with 70% ethanol for spreader sterilization
-Ice bucket containing:
pUC-Kan1 or pUC-Kan2 (100 ng/µl in sterile ddH2O)
pBR322 (New England Biolabs, diluted to 100 ng/µl in sterile ddH20)
29 µl
29 µl
D. M. Dean and J. A. Wilder
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10X FastDigest Green Buffer (“EB”; Fermentas)
18 µl
FastDigest EcoRI (“E”; Fermentas, #FD0274)
4 µl
FastDigest HindIII (“H”; Fermentas, #FD0504)
4 µl
10X T4 DNA ligase buffer (“LigB”; Fermentas)
3 µl
2 tubes of competent cells (“CC”)
100 µl/tube
Positive control plasmid (for “F” or “G” controls)
5 µl
Note: only 40% of the lab groups prepare either F or G.
Other groups prepare negative controls C, D, or E from their
pUC-Kan1, pUC-Kan2, or pBR322 plasmids.
-Microfuge tube rack containing:
Sterile water (“ddH2O”; autoclaved before use)
Luria broth (“LB”; prepared from LB powder, Fisher Scientific)
138 µl
1 ml
9 empty 1.5 ml microfuge tubes
Spin column from DNA Clean and Concentrator-5 Kit (Zymo Research)
Each group of 4 students receives:
-EASY CAST Electrophoresis System (Owl Scientific, Inc., Model #B1A)
-minigel comb with 12 teeth
-covered Erlenmeyer flask containing 60 ml of molten 0.8% agarose in 1X TBE + 0.5
µg/ml ethidium bromide
-2 plastic containers or weigh boats to hold the gel and gel comb
-10 ul Molecular Weight Standard (“MWS”; see SOLUTIONS below for formulation)
Each group of 8 students (one lab bench) receives:
-1 L of 1X TBE buffer containing 0.5 µg/ml ethidium bromide
-Minicell Power Supply (EC-103, EC Apparatus Corporation)
-Microcentrifuge with capacity to hold 24 microfuge tubes
-Empty microcentrifuge tubes, spin columns, and a supply of water for
counterbalancing microcentrifuge samples
-Scissors
-Roll of Parafilm
D. M. Dean and J. A. Wilder
Each class of 14-24 students shares:
-Water bath or dry bath set at 37°C (holding capacity of 5 microcentrifuge tubes
per student pair)
-Water bath set at 50°C for molten agarose gel flasks
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D. M. Dean and J. A. Wilder
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-DNA Clean and Concentrator-5 Kit (Zymo Research)
Note: we recommend that each kit solution be dispensed into three or more
aliquots per class section to avoid waiting lines and to minimize solution
contamination.
-UV transilluminator (FOTO/UV 15, Fotodyne)
-Gel camera (FOTO/Analyst Minivisionary, Fotodyne)
-T4 DNA ligase (Fermentas #EL0011; 1 µl/student pair, stored in -20°C freezer until
use)
-Dry incubator set at 37°C
-Boxes of latex or nitrile gloves
WEEK 1, DAY 3
Set up 2 of the following stations for the entire class:
-Fine-tipped, permanent marker to label culture tubes
-Masking tape
-Bunsen burner or ethanol lamp
-Lighter or matches
-Tungsten wire loop for picking colonies
-Beaker filled with 70% ethanol for loop sterilization
- LB-agar/kanamycin/tetracycline/X-gal plates containing transformants of pUC-Kan1
and pUC-Kan2-derived
Final Plasmids
-Scissors
-Roll of Parafilm
-Latex or nitrile gloves
Culture media
In a refrigerator near the lab station:
-Rack with 5 ml aliquots of LB/kanamycin/tetracycline in culture tubes (one culture
tube/student pair)
-A separate, labeled, empty culture tube rack for students to place their
inoculated culture tubes
D. M. Dean and J. A. Wilder
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WEEK 2
Each pair of students receives:
-A set of three Pipetman micropipettors (Gilson; P1000, P200, and P20) with
compatible tips
-Fine-tipped, permanent marker to label Eppendorf tubes
-Vortex Mixer (Fisher Scientific)
-Their inoculated culture from WEEK1, DAY 3, or backup culture if their culture is
not saturated
-Ice bucket containing:
10X FastDigest Green Buffer (“EB”; Fermentas)
6 µl
FastDigest BamHI (“B”; Fermentas, #FD0054)
1 µl
FastDigest HindIII (“H”; Fermentas, #FD0504)
1 µl
-Microfuge tube rack containing:
Sterile water (“ddH2O”; autoclaved before use)
1160 µl
8 empty 1.5 ml microfuge tubes
Spin column from Qiaprep Spin Miniprep Kit (Qiagen)
2 tubes compatible with class PCR machine
Each group of 8 students (one lab bench) receives:
-Microcentrifuge with capacity to hold 24 microfuge tubes
-Empty microcentrifuge tubes, spin columns, and a supply of water for
counterbalancing microcentrifuge samples
-Ice bucket containing:
5X GoTaq Flexi Green Buffer (“PCR buffer”; Promega)
50 µl
25 mM MgCl2 (Promega)
20 µl
10 mM PCR Nucleotide Mix (“dNTP mix”; Promega)
4 µl
16 µM pUC1 primer
12.5 µl
16 µM pUC2 primer
12.5 µl
16 µM Tet primer
Each class of 14-24 students shares:
12.5 µl
D. M. Dean and J. A. Wilder
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-Water bath or dry bath set at 37°C (holding capacity of 3 microcentrifuge tubes
per student pair)
-Water bath or dry bath set at 70°C (holding capacity of 3 microcentrifuge tubes
per student pair)
- Qiaprep Spin Miniprep Kit (Qiagen)
Note: we recommend that each kit solution be dispensed into three or more
aliquots per class section to avoid waiting lines and to minimize solution
contamination.
- GoTaq Flexi DNA Polymerase (Promega; 2 µl/group of
8 students, stored in -20°C
freezer until use)
-Automated PCR machine
-Boxes of latex or nitrile gloves
PRELABORATORY EXERCISE 2
Students are to complete this exercise before Week 3. As with PRELABORATORY
EXERCISE 1, they will need their laboratory manual (FILE S1) and a computer with
internet access.
WEEK 3
Each pair of students receives:
-A Pipetman micropipettor (Gilson; P20) with compatible tips
-Fine-tipped, permanent marker
-Vortex Mixer (Fisher Scientific)
-Microfuge tube rack containing their Control, B dig, H dig, PCR1 and PCR2 from
WEEK 2
Each group of 4 students receives:
-EASY CAST Electrophoresis System (Owl Scientific, Inc., Model #B1A)
-minigel comb with 12 teeth
-covered Erlenmeyer flask containing 60 ml of molten 0.8% agarose in 1X TBE + 0.5
µg/ml ethidium bromide
-2 plastic containers or weigh boats to hold the gel and gel comb
D. M. Dean and J. A. Wilder
-10 ul Molecular Weight Standard (“MWS”; see SOLUTIONS below for formulation)
Each group of 8 students (one lab bench) receives:
-1 L of 1X TBE buffer containing 0.5 µg/ml ethidium bromide
-Minicell Power Supply (EC-103, EC Apparatus Corporation)
-Microcentrifuge with capacity to hold 24 microfuge tubes
Each class of 14-24 students shares:
-Water bath or dry bath set at 37°C (holding capacity of 3 microcentrifuge tubes
per student pair)
-Water bath set at 50°C for molten agarose gel flasks
-UV transilluminator (FOTO/UV 15, Fotodyne)
-Gel camera (FOTO/Analyst Minivisionary, Fotodyne)
-Boxes of latex or nitrile gloves
SOLUTIONS
LB and LB-agar
Purchased in powder form from Fisher Scientific and prepared as described by
the manufacturer.
Antibiotics/X-gal
-Ampicillin sodium salt (Fisher Scientific, #BP1760-25)
100 mg/ml stock dissolved in ddH2O, sterile filtered, stored at -20°C for up
to a month. Dilute to
100 μg/ml (1:1000) in LB or LB-agar before use, after cooling autoclaved
solution below 60°C.
-Kanamycin sulfate
10 mg/ml stock dissolved in ddH2O, sterile filtered, stored at -20°C for up
to a month. Dilute to
50 μg/ml (1:200) in LB or LB-agar before use, after cooling autoclaved
solution below 60°C.
-Tetracycline hydrochloride
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D. M. Dean and J. A. Wilder
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12.5 mg/ml stock dissolved in 50% ethanol, no filtering, stored at -20°C for
up to a month.
Dilute to 12.5 µg/ml (1:1000) in LB or LB-agar before use after cooling
autoclaved solution below 60°C.
-X-gal (American Biorganics, #02-43805)
Stock 20 mg/ml stock dissolved in dimethylformamide, no filtering, stored at
-20°C for up to a month.
Dilute to 60 μg/ml (3:1000) in LB or LB-agar before use after cooling
autoclaved solution below 60°C.
10X TBE buffer (1L)
Use a 1000ml bottle with 500ml ddH2O and add:
108g Trizma base
55g Boric acid
40mls 0.5M EDTA (pH 8.0)
Bring final volume to 1000ml.
Ethidium bromide stock
Wearing gloves, dissolve 10 mg/ml ethidium bromide in ddH2O. Store in a glass
bottle covered with aluminum foil for protection from light.
1X TBE buffer
Dilute 10X TBE to 1X. To prepare gel running buffer, add ethidium bromide stock
to 0.5 µg/ml (1:20000 dilution). We have reused this buffer up to 4 times over
Week 1, but make a fresh solution for use during Week 3.
0.8% agarose gel
Prepare shortly before class: in a 125 ml Erlenmeyer flask, dissolve 0.48 g
agarose in 60 ml 1X TBE by heating to near boiling with stirring. Cool to about
60°C and add ethidium bromide stock to 0.5 µg/ml (1:20000 dilution). Cover the
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flask and store in 50°C water bath.
Molecular Weight Standard (“MWS”, prepare 10 ul per gel)
1 kB DNA ladder (Invitrogen #10787-018)
Sterile ddH2O
10X FastDigest Green Buffer (Fermentas)
3 ul
6 ul
1 ul