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Basics of Molecular Cloning:
Instructor's Manual
Purpose and Concepts Covered .1
This instructor's ! manual is avaliable
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Purpose and Concepts Covered
This introductory cloning laboratory is for use in courses that cover basic topics in molec-ular biology and genetics. Molecular cloning is a basic technique used in a molecularbiology labs. In this manual, we include a protocol for isolating the luciferase gene fromDNA using restriction digestion and cloning it into the multiple cloning region of a vector.
The cloned luciferase gene is then expressed in E. coli.
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Protocol for Cloning the Luciferase Gene into the pGEM® Vector
Preparation for the Laboratory
pGEM®-luc DNA (Cat.# E1541) pGEM®-4Z Vector (Cat.# P2161) BamHI restriction enzyme (Cat.# R6021; 10u/µl) SacI restriction enzyme (Cat.# R6061; 10u/µl) Buffer E (supplied with BamHI enzyme) Acetylated BSA (supplied with restriction enzymes) ! is a carcinogen.
Agarose, LMP, Preparative Grade for Large Fragments (>1,000bp; Cat.# V2831) Be sure to follow your TBE Buffer, 10X, Molecular Biology Grade (Cat.# V4251; or have students make institution's safety and their own buffer) disposal instructions Ethidium Bromide Solution, Molecular Biology Grade (Cat.# H5041) when working with Note: The gel fragment cleanup system will not work with DNA that
ethidium bromide.
has been stained with methylene blue.
BenchTop pGEM® DNA Markers (Cat.# G7521) Wizard® SV Gel and PCR Clean-Up System (Cat.# A9281) Blue/Orange Loading Dye, 6X (Cat.# G1881) T4 DNA Ligase (Cat.# M1801) agarose gel electrophoresis apparatus and power supply pipettors (0.5–10µl and 10–200µl) and appropriate sterile pipet tips long-wave UV light box and camera or scanner 1.5ml sterile tubes 2.A. Excising the Luciferase Gene from Surrounding DNA
In this part of the protocol, you will be isolating the gene of interest, the fireflyluciferase gene, from surrounding DNA. To remove the luciferase gene, you will beperforming a restriction digest using BamHI and SacI. You also will need to performthe same double digest on your target cloning vector (pGEM®-4Z Vector).
You will need to set up your restriction digest in a total volume of 20µl using the
guidelines below:
Restriction Enzyme Buffer E, final concentration 1X2µg of Acetylated BSA1µg of DNANuclease-Free Water to a volume of 19.0µl Mix these reagents by pipetting and then add: 5 units of BamHI in a total of 0.5µl5 units of SacI in a total 0f 0.5µl Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Telephone 608-274-4330 · Fax 608-277-2516 · www.promega.com
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1. Calculate the volume of Buffer E you will need in the reaction to achieve a final concentration of 1X.
Note to the instructor: Buffer E is supplied as a 10X solution. The students will
need to dilute the Buffer E so that its final concentration in the reaction is 1X.
(y µl) (10X Buffer E) = (20µl of 1X Buffer E)10y = 20y = 2µl of 10X Buffer E 2. Calculate the volume of DNA you will need to add to your reaction in order to digest 1µg.
Note to the instructor: The concentration of the pGEM®-luc Vector is
provided on the lot-specific product information sheet that accompanies the
vector. Using that information, your students can calculate the volume of
DNA to add to their reaction.
They can do the same for the target vector (pGEM®-4Z Vector).
Calculate the volume of Acetylated BSA you will need to add to your reac-tions, so that you have a total of 2µg of Acetylated BSA in your reaction. TheAcetylated BSA is supplied at a concentration of 10µg/µl.
Note: Pipetting volumes smaller than 0.5µl is very difficult. When determin-
ing how much Acetylated BSA to add, you may need to dilute the BSA to a
lower concentration so that you can pipette a larger volume into your reac-
tion (0.5 or 1.0µl).
Calculate the volume of water to add to bring the volume (without enzyme)to a total of 19.0µl. Enter the volumes of each component into the tablebelow: Volume to Add
Nuclease-Free Water
(add to a total volume of 19.0µl)
Restriction Enzyme Buffer E
Add the components, in the order listed, to a sterile 1.5ml tube. Mix them bypipetting.
Calculate the amount of each enzyme to add to your reaction. You will needto add a total of 5 units of each enzyme. Using the concentration of each ofthe enzymes provided to you by your instructor, determine what dilution (ifany) you need to make so that 0.5µl of the enzyme contains 5 units.
Note: High concentrations of either glycerol or enzyme can interfere with
your restriction digestion. Therefore, be sure that you do not add more than
1.0µl total of enzymes to your digest.
Add 5 units of BamHI and 5 units of SacI to your restriction digest. Mix gently by pipetting.
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Close the tube and centrifuge for a few seconds in a microcentrifuge to pullall of the liquid to the bottom of the tube.
9. Incubate at 37°C for 1 hour.
Note to the students: You should have performed two restriction diges-
tions: one of pGEM®-luc DNA to remove the luciferase gene insert, and one
of the pGEM®-4Z Vector to prepare it to receive the insert.
2.B. Purifying the Gene Fragment
After your restriction digest is complete, you will separate the luciferase gene frag-ment ( 1,740bp) from the remainder of the DNA using agarose gel electrophoresis.
The luciferase gene DNA can be purified from the gel using a commercially availableDNA cleanup system. You also will need to gel purify your restricted target vector(pGEM®-4Z Vector), which will be a fragment of 2,733bp.
Determine the appropriate agarose gel composition to use to isolate the
luciferase gene from the other DNA in the restriction digest using the table
provided at: www.promega.com/techserv/techref/agarose_polyacryl.htm
(Gel percentages: resolution of linear DNA on agarose gels). You will need
to clearly separate the 1,740bp gene fragment from the other fragment that
will be created by the digest (3,191bp).
Note to the instructor: We recommend 1.5% agarose.
2. Prepare 100ml of 10X TBE Gel Running Buffer using the following when working over aUV light box.
0.89M Tris base0.89M boric acid20mM EDTA (pH 8.0) Notes to the instructor:
Molecular Weight (MW) Tris base 121.14g/mol; MW boric acid 61.83g/mol Recipe for 1L 10X TBE from promega.com: Dissolve 108g of Tris base and55g of boric acid in 900ml of deionized water. Add 40ml of 0.5M EDTA (pH8.0), and increase the final volume to 1 liter. Store at room temperature or4°C.
Make 1X TBE by diluting the 10X TBE with water. Determine the final vol-ume of 1X TBE needed. Caluculate how much 10X TBE and how muchwater to mix to make this volume of 1X TBE.
Prepare your agarose gel 1.5% weight/volume agarose in 1X TBE (e.g.,1.5g of agarose in 100ml of 1X TBE). Put the agarose and the buffer in aflask that is at least twice the volume of buffer used. Heat the buffer andagarose in a microwave oven or on a hot plate to melt the agarose. (Do notlet the buffer boil over. The loss of buffer will change the agarose percent-age.) Allow the solution to cool to 50°C (cool enough to hold comfortably inyour hand) before pouring the solution into the gel tray. Add ethidium bro-mide to a final concentration of 5µg/ml to the cooling solution, and swirl theflask to mix. Remember to put in the comb to create loading wells before theagarose solidifies.
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2.B. Purifying the Gene Fragment (continued)
Notes to the instructor:
You will need to provide detailed instructions (volume, inserting comb, etc.)for gel preparation based on the particular gel aparatus you are using. We recommend adding ethidium bromide (final concentration 5µg/ml) to thegel before it is poured rather than using it in the gel running buffer. Althoughthe sizing is more accurate when the gel is stained after running, confiningthe ethidum bromide to the gel itself helps manage disposal.
When the gel has solidified, remove the comb, being careful not to tear thewells. Place the gel in the gel box and cover with 1X TBE buffer.
Remove 5µl from each of your restriction digests. Add 1µl of 6X blue/orangeloading dye. Add 1µl of dye to 5µl of the uncut DNA. 6. Load digests and run the gel until the markers in the loading dye have migrated to the appropriate point in the gel to have separated the fragments
in which you are interested. (See reference material at:
www.promega.com/techserv/techref/agarose_polyacryl.htm for help
determining this).
7. Turn off the electricity flowing to the gel box. Remove the gel, and place it on a long-wave UV box. (Short wave UV can damage the DNA.) Using ascalpel or razor blade, cut the 1,740bp gene fragment from the gel, andplace it in a preweighed 1.5ml microcentrifuge tube.
Weigh the tube with the gel slice, and determine the weight of your gel slice.
Note: At this point you will use the Wizard® SV Gel and PCR Clean-Up
System (Cat.# A9281) to extract your insert DNA from the gel slice. Detailed
instructions are available in Technical Bulletin #TB308, available at:
www.promega.com/tbs/
Add 10µl of Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 50–65°C for 10 minutes or until gel slice is completely dissolved.
10. Insert the SV Minicolumn into a Collection Tube from the Wizard® SV Gel and PCR Clean-Up System.
11. Transfer the dissolved gel mixture to the Minicolumn assembly. Incubate at room temperature for 1 minute.
12. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
13. Add 700µl of Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn intoCollection Tube.
Note: You can have students calculate the RPM of the centrifuge to achieve
a target RCF (in this case the target is 16,000 × g) for your
particular microcentrifuge using the radius of the rotor. There is an online
calculator at: www.sciencegateway.org/tools/rotor.htm, but if you
prefer, your students can use this equation:
g = (1.118 × 10-5) RS2 Where g is the relative centrifugal force, R is the radius of the rotor in cen-timeters, and S is the speed of the centrifuge in revolutions per minute.
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Note: The Wizard® SV
14. Repeat Step 13 with of 500µl Membrane Wash Solution. Centrifuge at Gel and PCR Clean-Up 16,000 × g for 5 minutes.
System recommends 15. Empty the Collection Tube, place the open column in a stand on the lab spinning down the mini-column for one minute bench to evaporate the ethanol. There should be no longer an odor of the to help with ethanol Column Wash buffer.
evaporation. We elimi- 16. Carefully transfer Minicolumn to a clean 1.5ml microcentrifuge tube.
nated this direction fromStep 15 because of 17. Add 50µl of Nuclease-Free Water to the Minicolumn. Incubate at room safety concerns.
temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute.
Instructors should usetheir discretion about 18. Discard Minicolumn and store DNA at 4°C or –20°C.
recommending this pro-cedure for students.
2.C. Ligating the Insert into the Vector
Determining the Vector:Insert Ratio Note: To estimate DNA
After the insert DNA has been prepared for ligation, estimate the concentration by concentration, students comparing the staining intensity with that of DNA molecular weight standards of should prepare a sec- known concentrations on an ethidium bromide-stained agarose gel. If the vector ond gel and load it with DNA concentration is unknown, estimate the vector concentration by the same 10µl of the digests and method. Test various vector:insert DNA ratios to determine the optimal ratio for a 500ng of Lambda Hind particular vector and insert. In most cases, either a 1:1 or a 1:3 molar ratio of vec- IIIDNA markers to esti- tor:insert works well. The following example illustrates the calculation of the amount mate the concentration.
Alternatively this can be of insert required at a specific molar ratio of vector. done using a spectrom- [ng of vector × size of insert (in kb)] ÷ size of vector (in kb) × molar amount of eter if one is available.
(insert ÷ vector) = ng of insert Example: How much 500bp insert DNA needs to be added to 100ng of 3.0kb vectorin a ligation reaction for a desired vector:insert ratio of 1:3? [(100ng vector × 0.5kb insert) ÷ 3.0kb vector] × (3 ÷ 1) = 50ng insert Briefly centrifuge the pGEM®-4Z Vector and the DNA insert tubes to collect
contents at the bottom of the tube. (Note: Detailed information about the
vector is provided in Technical Bulletin #TB033, available at:
www.promega.com/tbs .)
Set up ligation reactions as described below. Vortex the T4 DNA Ligase 10XBuffer vigorously before each use. Use 0.5ml tubes known to have low DNA-binding capacity.
T4 DNA Ligase 10X Buffer T4 DNA Ligase (3 Weiss units/µl) Nuclease-Free Water to a volume of Note: If digest yields are low, it may be necessary to increase the overall
! volume of the reaction. Remember to maintain rations of volume is
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2.C. Ligating the Insert into the Vector (continued)
Mix the reactions by pipetting. Incubate the reactions for 1 hour at roomtemperature. Alternatively, incubate the reactions overnight at 4°C for themaximum number of transformants. Note to the Instructor: Because the target vector was cut with two different
enzymes that do not generate compatible ends, the vector should not spon-
taneously religate. You might discuss with the students what precautions
they would have to take if the enzymes produced "compatible ends" or if
they were only cutting the vector with a single enzyme (i.e., removing phos-
phate groups from the end of the vector so that the ligation reaction could
not be catalyzed between the two ends).
2.D. Transforming Bacteria with Your Clone
Single-Step KRX Competent Cells (Cat.# L3002; three tubes per lab group) vector with ligated insert uncut vector positive transformation control cut vector negative transformation control 17 × 100mm polypropylene tubes (BD Falcon Cat.# 352059) or 1.5ml microcentrifuge tubes 37°C shaking water bath or incubator pipettors (0.5–10µl and 10–200µl) and appropriate sterile pipet tips glass "hockey stick" for plating cells on selective plates Prepare LB/ampicillin/IPTG/X-Gal plates (see Section ).
Centrifuge the ligation reactions briefly. Place the high-efficiency Single-Step KRX Competent Cells in an ice bathuntil just thawed (5 minutes). Mix cells by gently flicking the tube.
Add the 2µl of the ligation reaction to one tube of thawed cells. Add 0.1ng ofuncut plasmid to a second tube of thawed cells for the positive control tube.
Add 0.1ng of cut plasmid to a third tube of thawed cells for the negative con-trol. Gently flick the tubes, and incubate on ice for 5 minutes.
5. Heat-shock the cells for 15–20 seconds in a water bath at exactly 42°C. DO NOT SHAKE. Immediately return the tubes to ice for 2 minutes.
6. Add 950µl of room temperature SOC medium to the ligation reaction trans- formations and 900µl to the tranformation control tube. Incubate for 1.5 hours at 37°C with shaking ( 150rpm).
Plate 100µl of each transformation culture onto duplicateLB/ampicillin/IPTG/X-Gal plates. For the transformation control, a 1:10 dilution with SOC is recommended prior to plating.
Incubate plates overnight at 37°C. Select white colonies.
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2.D. Transforming Bacteria with Your Clone (continued)
Calculation of Transformation Efficiency
For every transformation with competent cells, we recommend performing a transfor-mation control experiment using a known quantity of a purified, supercoiled plasmidDNA (e.g., pGEM®-4Z Vector, Cat.# P2161). Calculate the transformation efficiencyas described below.
transformation efficiency (cfu/µg) = (cfu on control plate ÷ ng of supercoiled vectorplated) × (103ng/µg) × final dilution factor cfu = colony forming units A 100µl aliquot of competent cells is transformed with 1ng of supercoiled pGEM®-4ZVector DNA. Ten microliters of the transformation reaction (0.1ng total DNA) isadded to 990µl of SOC medium (1:100 dilution). Of that volume (1,000µl), a 100µlaliquot is plated (1:1,000 final dilution), and 100 colonies are obtained on the plate.
What is the transformation efficiency? (100cfu ÷ 0.1ng of supercoiled vector plated) × (103ng/µg) × 1,000 = 1 x 109 cfu/µg Note to the Instructor: If you have your students plate 10µl, 100µl, 500µl of the
uncut positive transformation control, they can practice calculating the transforma-
tion frequency for their experiment using the above equation.
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2.E. Expressing Luciferase in E. coli
Terrific Broth + ampicillin (100µg/ml) (fresh) Luria Broth + ampicillin (100µg/ml) (fresh) 20% solution of Rhamnose (L-rhamnose Monohydrate, Cat.# L5701) 37°C shaking incubator for liquid culture 15–25°C shaking incubator for liquid culture Luciferase Assay Reagent (LAR; Cat.# E1483) 450µl 100mM sodium citrate (pH 5.5) pipettors (0.5–10µl, 10–200µl and 1ml) and appropriate sterile pipet tips White colonies should have the luciferase gene cloned in the correct orientation inthe pGEM®-4Z Vector to be expressed from the T7 promoter on the vector. Since theKRX strain of E. coli contains the necessary information to express a gene from theT7 promoter, your students should be able to see "glowing" cells when the cells areprovided with the appropriate substrates for the luciferase reaction.
From your transformation plates, use a sterile innoculating loop to pick fouror five white colonies into indivdual 5ml liquid cultures of LB Broth + Ampusing aseptic technique and grow overnight at 37°C with shaking*. (Sealyour original plates and store them, inverted at 4°C.) Note: Better growth was observed when culture tubes were incubated at an
angle during overnight shaking.
Dilute your overnight cultures 1:100 into new, labeled tubes of Terrific Broth+ Ampicillin. Grow cultures at 37°C with shaking at 275rpm until they reachan O.D.600 of 0.8–1.0.
Shift cultures to a second incubator shaker set at 15–25°C and continueshaking until cells reach an O.D.600 of 1.0–1.5.
Induce protein expression by adding rhamnose to a concentration of 0.1%.
(1:200 dilution of a 20% rhamnose solution).
Grow cultures overnight at 15–25°C with shaking at 275rpm. Transfer 500µl of each your cultures to labeled 1.5ml centrifuge tubes. Pelletthe cells by centrifugation at 10,000 × g for 3 minutes in a microcentrifuge.
Aspirate the supernatant and resuspend the cell pellet in 450µl 100mMsodium citrate + 50µl Luciferase Assay Reagent.
Take the tube into a dark room. Vigorously shake the tube to break up theclumps of cells and to introduce oxygen into the reaction.
8. Are any of the cells in your tubes "glowing"? Note: The luminescence will be very faint to the naked eye. Comparison to
blank tubes helps with visual dectection of luminescence.
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Composition of Buffers and Solutions
IPTG stock solution, 0.1M
SOC medium
1.2g IPTG (Cat.# V3955) 2.0g Bacto®-tryptone Add water to 50ml final volume. Sterilize through a 0.2µm 0.5g Bacto®-yeast extract filter unit, and store at 4°C for up to 60 days or store in aliquots at –20°C for up to six months.
1ml Mg2+ stock solution, 2M LB medium with or without antibiotic
1ml 2M glucose, filter-sterilized 10g/L Bacto®-tryptone Add Bacto®-tryptone, Bacto®-yeast extract, NaCl and KCl 5g/L Bacto®-yeast extract to 97ml distilled water. Stir to dissolve. Autoclave, and cool to room temperature. Add sterile 2M Mg2+ stock and 2M Adjust the pH to 7.5 with NaOH. Autoclave to sterilize.
glucose stock, each to a final concentration 20mM. Bring to Allow the auto claved medium to cool to 55°C, and add 100ml with distilled water. Filter through a sterile 0.2µm fil- antibiotic to a final concentration shown in the table below.
Antibiotics Useful for Plasmid Selection in KRX.
12.0g Bacto®-tryptone 24.0g Bacto®-yeast extract 100ml potassium phosphate, 0.89M Add Bacto®-tryptone, Bacto®-yeast extract, glycerol to 750ml distilled water. Stir to dissolve, then bring the vol- ume to 900ml with distilled water. Autoclave, and cool to60ºC. Add 100ml of sterile 0.89M potassium phosphate to a final volume of 1,000ml. Filter through a sterile 0.2µm unit.
For LB plates, include 15g agar prior to autoclaving.
Available from Promega (Cat.# V3941) at a concentration of50mg/ml in dimethylformamide.
rhamnose, 20% (w/v)
10g L-rhamnose monohydrate Note for all filter sterilized solutions: Filter-sterilizing
Add distilled water to 45ml, sterilize through a 0.2µm filter units should be prerinsed with distilled water before use to unit and store in aliquots at –20°C.
remove any toxic material.
To prepare a sodium citrate solution at pH 5.5 you wouldweigh the appropriate amount of trisodium citrate dihy-drate solid (m.w. 294.12 g/L) depending the desired molar-ity. Dissolve it in deionized water and titrate the pH to 5.5using citric acid monohydrate (m.w. 210.14 g/L). Note:There are 3 types of sodium citrate salts: monosodium cit-rate, disodium citrate, and trisodium citrate. Disodium cit-rate and trisodium citrates are commonly used in the lab.
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