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Project Documentation & Protocols: Maize Mapping Project: RFLP Protocols
Disclaimer: Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by the University of Missouri-Columbia RFLP Laboratory, and also does not imply approval to the exclusion of other products that may also be suitable.
The following procedures represent those currently in use or those which have been used successfully in the Maize RFLP Laboratory at the University of Missouri-Columbia. While there are probably as many ways to isolate plant genomic DNA and to transfer DNA from a gel to a membrane as there are researchers in the area, these procedures have been used routinely and appear to be very dependable in the RFLP analysis of the various plant species studied in the lab. While these procedures basically are those in use as of the time of this revision, we are constantly evaluating various modifications to each of the steps, particularly if they would result in significant cost or time savings. Since the lab data acquisition phase of RFLP research is simply large scale molecular biology, most of these procedures were drawn from those published in the literature, from those presented in the various molecular biology manuals, and from those sent to us by fellow RFLP researchers and molecular biologists. We have made an effort to cite the source or contributor for each of the procedures, but we acknowledge that many individuals may have contributed to the refinement of each of the steps. We greatly appreciate all of the ideas, comments and suggestions offered by the many visitors to the lab. We also look forward to future interactions with all of those interested in RFLP research. While every effort has been made to make the procedures and the various solutions used in each step as accurate as possible, it still would be wise to check each of the steps and corresponding solutions for accuracy. If any mistakes are found, or you have any comments or suggestions, we would greatly appreciate receiving them. Please address your comments or questions and requests for copies of this manual to Theresa Musket, Missouri Maize Project, 403 Tucker Hall, University of Missouri-Columbia, Columbia, Missouri 65211, (573) 8843715, musket@teosinte.agron.missouri.edu. Safety: An effort has been made to provide safety precautions pertaining to all procedures that are applicable. Individuals should be properly trained in the safe handling of all hazardous, biological, and radiation chemicals, reagents, and procedures. All hazardous and radiation safety procedures should be followed in accordance with your own individual institution. Good Luck in your research endeavors and Happy Mapping! Correct Citation: Gardiner, J. M., 1998. UMC Maize RFLP Procedures Manual. Unpublished. (A compilation of procedures with modifications, updated 1998). 1. Harvest leaves from plant. It is preferable to use young leaves, or at least older leaves without necrotic areas or lesions. 2. Place leaves on ice during harvest to make sure that they are kept cool. Remove the midrib in older leaves. Leaves may be wrapped in foil or preferably in a fiberglass screen mesh bag. 3. For immediate lyophilization: Place leaf sample in a styrofoam container or some other type of container able to hold liquid nitrogen. Quick-freeze samples in liquid nitrogen. Once frozen do not allow samples to thaw until dried! 4. Transfer frozen leaf samples to lyophilizer. Make sure that the lyophilizer is down to temperature (the chamber is -60.C at least) and pulling a good vacuum (<10 microns) before loading samples. Do not overload lyophilizer: make sure vacuum is always <100 microns and condenser temperature is <-60.C. Samples should be dried in 72 hours. Typically, fresh weight approximately 10 x dry weight.
5. Dried leaf samples may be stored in sealed plastic bags at room temperature for short periods of time (<1 month) or at -20.C for several years. Desiccant should be included in the plastic bag to prevent the samples from taking on water. Safety: The grinder should be set up in a fume hood to help carry off the particulate matter generated. Even with a hood, a respirator should be worn that has a high efficiency dust/fume/mist cartridge, a 'nuisance-dust' mask is not enough. Safety glasses and hearing protection are also recommended.
1 Cyclone sample mill(Based on method of Saghai-Maroof, et al., 1984, PNAS 81:8014-8018) Safety: Wear a lab coat, latex gloves and safety glasses. All phenol and chloroform extractions should be performed in a fume hood. If gloves become contaminated with phenol or chloroform, change them immediately. Plant extracts containing CTAB, phenol, chloroform or any alcohols should be saved for proper disposal. Coordinate disposal with the regulations of individual work places. 1. Add 9.0 ml of 65.C CTAB Extraction Buffer to 300-400 mg ground, lyophilized tissue, in a 15 ml polypropylene centrifuge tube. It is best to distribute tissue along the sides of the tube before adding buffer, to avoid clumping of dry tissue in the bottom. Mix several times by gentle inversion1. 2. Incubate for 60-90 min, with continuous gentle rocking in a 65.C oven. Do not exceed 90 minutes as DNA yield will be compromised. 3. Remove tubes from oven, wait 4-5 min to prevent spattering, then add 4.5 ml chloroform/octanol (24:1). Rock gently to mix for 5 - 10 min (recommend 10 min). 4. Spin in a table-top centrifuge2 for 10 min at 2000 x g at room temperature (RT). 5. Pour off top aqueous layer into new 15 ml tubes. Add 4.5 ml chloroform/octanol and rock gently for 5-10 min (recommend 10 min). 6. Spin in a table-top centrifuge2 for 10 min at 2000 x g at RT. 7. Pipette off top aqueous layer into new 15 ml tubes. Add 50 µl of 10 mg/ml RNAse A (pre-boiled). Mix by gentle inversion and incubate for 30 min at 37oC or room temperature. 8. Add 6.0 ml ice-cold isopropanol (2-propanol). Mix by gentle inversion. 9. Remove precipitated DNA with sterile glass hook. 10. Place hook with DNA in 5 ml plastic tube containing 1 ml of TE pH 8.0 . Rock gently overnight at room temperature to disperse DNA. 11. Phenol extract each sample with 1 ml (1x original TE volume) of equilibrated phenol. Centrifuge the sample 10 min at 2000 x g in swinging bucket rotor. 12. Transfer top (aqueous) layer to new tube. Extract DNA with a 1 ml (1x original TE volume) of chloroform/octanol. Centrifuge the sample 10 min at 2000 x g in swinging bucket rotor2. Transfer top (aqueous) layer to new tube. 13. Precipitate DNA by adding 50 µl of 5 M NaCl and then 2.5 ml absolute EtOH (2.5x original TE volume), mix by gentle inversion. 14. Remove precipitated DNA with sterile glass hook. Place hook with DNA in 5 ml plastic tube containing 3-4 ml of WASH 1. Leave DNA on hook in tube for about 20 min. 15. Rinse DNA on hook briefly in 1-2 ml of WASH 2 and transfer DNA to 5 ml plastic tube containing 0.3 ml TE pH 8.0 . Rock gently overnight at room temperature to disperse DNA. Store samples at 4.C. NOTES: DNA can be extracted from 1.5 g of dried tissue in 50 ml polypropylene tubes. Use 27 ml CTAB Extraction Buffer, 13.5 ml chloroform/octanol per extraction, incubate with 150 µl 10 mg/ml RNAse A, and precipitate with 18.0 ml isopropanol. Option A: Resuspend in a 15 ml polypropylene culture tube in 2 ml TE pH 8.0 , phenol and then chloroform/octanol extract with 2 ml of each. The extractions (phenol and then chloroform/octanol) may need to be repeated to sufficiently clean-up the sample. Option B: If the sample will not completely resuspend in 1 ml of TE pH 8.0 , the volume can be increased to a maximum of 4.0 ml. REMEMBER this will require proportional increases in the volume of 5 M NaCl and absolute EtOH added to precipitate the DNA. 1 Note that below 15oC the CTAB/nucleic acid complex may precipitate prematurely; this would ruin the preparation and could damage the centrifuge.2 3000-3200 rpm in Beckman Table-Top GP centrifuge with swinging bucket rotor (holds 56 x 15 ml tubes).
1 Make fresh before use.2 Add BME (ß-mercaptoethanol) to warmed buffer (60-65.C), just prior to use.3 CTAB = Mixed alkyltrimethyl-ammonium bromide (Sigma).
WASH 1: 76% EtOH, 0.2 M NaOAc
WASH 2: 76% EtOH, 10 mM NH4OAc
UV Quantification of DNA Add 5 µl of each DNA sample to 745 µl TE (dilution factor, d.f., = 150), read OD260 and OD280 to determine purity. Dilute samples to 0.4 µg/µl (or other concentration as needed) with TE. Store at 4.C for up to 6 months. Store at -20.C for long term storage. DNA concentration (µg/ µl) = [OD260 x 150 d.f. x 50 m g/ml]/1000 The ratio OD260/OD280 should be determined in order to assess the purity of the sample. If this ratio is approximately 1.65 to 2.0, the absorption is probably due to nucleic acids. A ratio of 1.6 or less indicates that there may be proteins and/or other UV absorbers in the sample, in which case it is advisable to reprecipitate the DNA. A ratio higher than 2.0 indicates the samples may be contaminated with chloroform, phenol, or RNA and should be reprecipitated with ethanol or RNAsed. A DNA Quantification Program for the Beckman DU-65 Spectrophotometer is included which provides automated sample entry (with sipper) and calculates all appropriate values for each sample. . Genomic DNA (based on method from T. Helentjaris, NPI)
REPRECIPITATION OF DIGESTED DNA: 1. Add 20 µl of 5 M NaCl to each 300 µl rxn. 2a. Add 750 µl (2.5 volumes) of EtOH, mix, place at -80.C for 30 min, then centrifuge in microfuge at full-speed (~12,000 rpm) for 5-7 min, just as samples thaw. or- 2b. Add 350 µl of ice-cold isopropanol, mix, then centrifuge in microfuge at full-speed for 5-7 minutes. 3. Pour off supernatant and invert tubes to air dry overnight on the bench. Avoid overdrying (overnight under vacuum) as this makes samples difficult to resuspend. 4. Dissolve pellet in the volume of TE pH 8.0 which is convenient for loading into wells of an agarose gel. Typically, 20 µl of TE pH 8.0 and 5 µl of 5X SGB per well is sufficient. Generally, pellets are dissolved in 2 - 3 hours. (Best to dissolve DNA in TE pH 8.0 first, then add 5X SGB.) Warming TE pH 8.0 to 65oC prior to addition will speed the process.
5X SGB (Sample Gel Buffer)
*Sodium Salt Store in refrigerator or freezer.
Neutral Agarose Gel Electrophoresis (based on method from T. Helentjaris, NPI) Safety: Electricity requires respect. Make sure gel box connections to power supplies are solid and protected. Replace frayed wires. Ethidium bromide is mutagenic, wear gloves when handling stock and any solution or gel that contains ethidium. Gels, gel buffers, tips and gloves that have ethidium bromide on them should be disposed of properly, according to the regulations of the work place. 1. Weigh agarose into 1X TAE Gel Buffer solution and mix. Boil agarose for 1 minute, then cool to 60.C.
2. Tape the ends of gel tray, pour agarose into tray and insert combs. Solidify 1 hour. 3. Remove tape and place tray in rig with 1X TAE Gel Buffer . Pour enough buffer into the gel rig to cover the gel by at least 0.5 cm, then remove combs and load samples containing 1X SGB into wells. 4. Electrophorese samples into gel at 100 mA until all dye has migrated into the gel, then electrophorese at 15-20 mA until blue dye has migrated the desired distance. 5. Remove tray from rig and stain in 1 µg/ml ethidium bromide (50 µl of 10 mg/ml ethidium bromide in 500 ml dH2O) for 20 min with gentle shaking. CAUTION: Ethidium bromide is extremely mutagenic - wear gloves when handling and use extra caution. 6. Rinse gel in dH2O for 20 min, slide gel onto a UV transilluminator and photograph. For Fotodyne PCM-10 camera with 20 x 26 cm hood and Type 667 Polaroid film, use an f8 with a 4 second exposure.
Adjust pH to 8.0 with Glacial Acetic Acid.
1X TAE Gel Buffer (Working Stock) Add 400 ml 50X TAE Gel Buffer into 19.6 liters dH20. Mix.
Molecular Weight Marker (32 pg / ml) 3.2 ml (Pharmacia # 27-4060-01) 10 ml 5X SGB 40 ml TE pH 8.0 Store at 4.C.
Safety: Electricity requires respect. Make sure gel box connections to power supplies are solid and protected. Replace frayed wires. Ethidium bromide is mutagenic, wear gloves when handling stock and any solution or gel that contains ethidium. Gels, gel buffers, tips and gloves that have ethidium bromide on them should be disposed of properly, according to the regulations of the work place. 1. Weigh agarose into 1X TAE buffer solution and mix. Boil agarose for 1 minute, then cool to 60.C.
2. Tape the ends of gel tray such that the tray will be able to accommodate 2 layers. For example with a 20 x 25 cm double gel, the first layer will have 350 ml and the second layer 325 ml. Pour the first layer of agarose into tray and insert combs. Allow to solidify for 1 hour. 3. Allow second gel solution to cool to 60.C and pour over first layer. Pour the solution slowly, gradually moving back and forth across the bottom end of the gel rig so as to avoid melting the bottom layer. Allow to solidify 1 hour. 4. Remove tape and place tray in rig. Pour enough 1X TAE Gel Buffer into the gel rig to cover the gel, then remove combs and load samples into the wells. Load the wells of the gel to the top of the second layer. It typically takes 50 to 60 µl to fill each well. 5. Electrophorese samples into gel at 100 mA until all dye has migrated into the gel, then electrophorese at 15-20 mA until blue dye has migrated the desired distance. 6. Remove tray from rig. Place the double thick gel in a large tray with 1X TAE Gel Buffer from the run. Split the gels by starting at the corner of the gel with a thin spatula or a small glass rod. Care will need to be taken not to break the gel along the wells. 7. Stain each gel in 1 µg/ml ethidium bromide (50 µl of 10 mg/ml ethidium bromide in 500 ml dH2O) for 20 min shaking gently. CAUTION: Ethidium bromide is extremely mutagenic - wear gloves when handling and use extra precaution. 8. Rinse gel in dH2O for 20 min, slide gel onto a UV transilluminator and photograph. For Fotodyne PCM-10 camera with 20 x 26 cm hood and Type 667 Polaroid film use an f8 with a 4 second exposure. NOTES: * (Bottom + Top Layer), respectively. (based on method from T. Helentjaris, NPI) Safety: For the protection of personnel and to assure the quality of the transfer, lab coats, latex gloves, and safety glasses should be worn. Membrane: MSI Magnacharge (Fisher # NB4HYA0010). If other membrane is to be used, follow manufacturers directions for blotting as they may vary from those described below. 1. Denature gel for 30 min in Denaturing Buffer : 0.4 N NaOH, 0.6 M NaCl. Gel can be flipped over at this point by placing it between two pieces of plexiglass and carefully flipping over. This will allow for a smoother surface to lay the membrane against when constructing the blot. 2. Neutralize gel for 30 min in Neutralizing Buffer: 0.5 M Tris-7.5, l.5 M NaCl. Construction of Wet Blot Transfer System 3. Place 4 small lids in a tray for transfer. Add transfer buffer up to top of lids. Briefly, dip 2 sheets of precut gel blotting paper 20 x 41 cm in transfer buffer and center over a piece of plexiglass. Lay plexiglass centered over 4 lids in tray. Curl ends of blotting paper in under plexiglass. This will act as a wick for the transfer buffer. Use a glass rod to smooth out any bubbles on blotting paper. 4. Place gel onto blotting paper with open-side of wells face-down. 5. Cut membrane to size of gel. Label using a Pigma pen (Sakura Color Products Corp.) or nick membrane before blotting, for later identification. Wet membrane in transfer buffer. Place cut piece of membrane on gel, label-side down to identify transfer side of membrane. Use a glass rod to smooth membrane on gel surface.
Important! Make sure that there are NO air bubbles between blotting paper, gel, and membrane. Use transfer buffer between each layer to avoid bubble problems.
Note: Paper towels should cover entire area of gel and not extend beyond edge of membrane. This will short-circuit the transfer.
Denaturing Buffer: 0.4 N NaOH, 0.6 N NaCl (1 liter/gel)
Neutralizing Buffer: 0.5 M Tris-7.5, 1.5 M NaCl (1 liter/gel)
Adjust pH to 7.5 with conc. HCl OR
Transfer Buffer: 25 mM NaPO4, pH 6.5 (4 liters/gel)
Oligolabeling (based on method of Feinberg & Vogelstein, 1984, Anal. Biochem. 137:266-267) Safety: This protocol requires special training on the part of the researcher because radioisotopes are incorporated into DNA to generate a probe. Follow the recommendations of the radiation safety office. In general, 32P work should be carried out in a designated radiation area behind a plexiglass shield while wearing a lab coat, safety glasses and at least one pair of latex gloves. Disposal of radioactive materials and solutions should be done with strict adherence to the regulations of the radiation safety office. Note: Label 1 Full Rxn of MW DNA for labeling (or l mix)/ 5 FULL RXNs of probe DNA. You will use 8-10 µl of labeled standard for each blot you hybridize. The basic oligolabeling reaction is as follows:
1. Add DNA (or MW DNA for labeling Mix , 10 µl of 5 ng/µl per FULL RXN) and ddH2O to 500 µl microfuge tube. Denature DNA by heating at 95.C for 5-7 min. Cool on ice. 2. Prepare a bulk reaction mix of OLB, BSA , dCTP, and Klenow* just before use (make-up enough reaction mix for the required number of labeling RXNs plus 2 more FULL RXNs) and aliquot appropriate amount into each tube containing DNA. Add 10 µl OLB reaction mix to 15 µl ddH2O as a control for the PEI Cellulose TLC. *DO NOT allow Klenow to stand at room temperature, remove required amount from freezer and immediately return to freezer, add to bulk mix just prior to use. 3. Incubate overnight at room temperature. (Minimum time = 4 hours) or in a 37oC water bath or heating block for 2 hours. Be sure to poke a hole in the top of the microfuge tube to prevent the top from popping open during denaturing or labeling. 4. Spot 0.5 µl aliquot onto PEI cellulose for incorporation check (see Checking Incorporation by PEI Cellulose TLC). 5. If desired, remove unincorporated dCT32P by spin column (we generally have not found this to be necessary). 6. Denature probe by heating at 95.C for 5-7 min. Cool on ice. Add to hybridizations.
OLB TE (3 mM Tris-HCl, 0.2 mM EDTA, pH 7.0) Add 300 µl of 1 M Tris -HCl and 40 µl of 0.5 M EDTA pH 8.0 to 90 ml of ddH2O. Bring pH to 7.0 with HCl and bring volume to 100 ml.
100 mM dATP (Pharmacia 27-5500-0) MW=589.2 Dissolve 10 mg in 169.7 µl of OLB TE (dissolve directly in original bottle). Store in 30 µl aliquots at -20.C. Mark tubes with black tops.
100 mM dGTP (Pharmacia 27-5700-0) MW=609.2 Dissolve 10 mg in 169.1 µl of OLB TE (dissolve directly in original bottle). Store in 30 µl aliquots at -20.C. Mark tubes with green tops.
100 mM dTTP (Pharmacia 27-5800-0) MW=584.1 Dissolve 10 mg in 171.5 µl of OLB TE (dissolve directly in original bottle). Store in 30 µl aliquots at -20.C. Mark tubes with red tops. Tris-MgCl Solution (1.25 M Tris-HCl, 0.125 M MgCl2, pH 8.0) Dissolve 19.7 g Tris-HCl and 2.54 gïMgCl2.6H2O in 75 ml ddH2O. Bring pH to 8.0 and bring volume to 100 ml.
To 1.0 ml Tris-MgCl Solution , add 18 µl 2-mercaptoethanol, 5 µl 100 mM dATP , 5 µl 100 mM dGTP , and 5 µl 100 mM dTTP . Store at -20.C for no more than 90 days. Mark tube with red and blue tops.
Dissolve 23.8 g of Hepes (Sigma #H3375, MW=238.3) in 35 ml ddH2O. Bring pH to 6.6 with 4 M NaOH. Bring final volume to 50 ml, recheck pH and correct if necessary. Store in l ml aliquots at -20.C. Mark tubes with black and blue tops.
OLB: (l.0 A : 2.5 B : 1.5 Hexamers) Add 1110 µl of OLB TE to the 50 Units of hexamers (Pharmacia 27-2166-01) in the shipping bottle, then add 738 µl of Solution A, and 1850 µl of Solution B. Store in 200 µl aliquots at -20.C. Mark tubes with red and black tops. - OR - To make smaller quantities of OLB: Mix 50 µl Solution A, 125 µl Solution B, and 75 µl Hexamer Solution. Store in 100 µl aliquots at -20.C. Mark tubes with red and black tops. Dissolve 50 units of hexamers (Pharmacia 27-2166-01) in 1110 µl of OLB TE . Store in 75 µl aliquots at -20.C. Mark tubes with green and black tops.
OLB Stop Mix
BSA (50 mg/ml) Purchase DNAse-free BSA from BRL (cat. #5561) or use the 1 mg/ml BSA provided by New England Biolabs with restriction endonucleases.
Purchase from BRL (# 18012-096).
Purchase from Dupont (NEN) (# BLU-513H).
l DNA for labeling (1.67 ng / ml) 3.2 ml l DNA (Pharmacia # 27-4060-01) 15 ml dH2O Store at 4.C.
Removal of
Unincorporated dNTP's (based on method from Maniatis) Safety: This protocol involves radiation. The same safety requirements for the oligolabeling protocol apply here. 1. Place small (2 cm2) piece of glass wool in bottom of 1 ml plastic syringe. Place syringe in 15 ml plastic centrifuge tube. 2. Fill syringe with Sephadex G-50 equilibrated in STE all the way to the top. Let beads settle for 5-10 min. 3. Add 20 µl denatured 10 mg/ml salmon sperm DNA to each tube. Let sit for 5 min. Denature salmon sperm DNA (SS DNA) by adding 1/10 volume of 1N NaOH. Vortex briefly and let sit for 10 min at RT. Neutralize by adding 1/10 original volume of l.8 M Tris-HCl, 0.2 M Tris-OH. Use desired original volume. -OR - Denature by heating at 95.C for 10 min followed by cooling on ice. 4. Centrifuge for 4 min at 1600 xg. Packed volume should be around 0.8 ml. 5. Transfer syringe to 15 ml glass centrifuge tube with 1.5 ml microfuge tube in bottom. Microfuge tube should be low enough to collect around 200 µl of volume without touching column. 6. Add probe (95 µl) to column and wash tube with additional 100 µl STE and add to column. Centrifuge as above. 7. Carefully remove and discard syringe. Remove and cap microfuge tube.
Checking Incorporation by PEI Cellulose TLC (based on method from W. Woodman, ISU) Safety: This protocol involves radiation. The same safety requirements for the oligolabeling protocol apply here. NOTE: We have found PEI Cellulose to be an inexpensive and reliable way of checking incorporation of dCTP into DNA. It is available from Sybron/Brinkman (Cat # 6610100-2, 20x20cm, 25 sheets). 1. Cut a piece of PEI Cellulose 6.0 cm high and wide enough to allow a sample to be spotted every 0.5 cm. 2. Draw a pencil line across the width at 1.0 cm from the bottom. 3. Spot 0.5 µl of each sample just above the line. Spot a sample of dCT32P of similar dilution as an internal standard. 4. After the samples have dried, place the chromatogram in an enclosed tank (or beaker with Petri dish cover). 5. Develop in 0.75 M NaPO4 pH 3.5 until front is within 1-5 mm of the top. For best results, prepare 0.75 M NaP04 with monobasic phosphate and pH to 3.5 with phosphoric acid. 6. Remove and let dry. Wrap in plastic wrap.
Prehybridizations and Hybridizations(based on method from T. Helentjaris, NPI) Safety: This protocol involves radiation. The same safety requirements for the oligolabeling protocol apply here. 1. Use 32 ml of 65oC prehybridization solution per 275 cm2 blot if hybridizing in seal-a-meal bags or 40 ml of prehybridization solution if using bottles or boxes. Seal bag after removing large air bubbles. Prehybridize at 65.C, slowly rocking, 4 - 5 hours for first-use blots and at least 2-3 hours for previously used blots. 2. Squeeze out as much of the prehybridization solution as possible from the bag and add 24 ml of 65oC hybridization solution per 275 cm2 blot.
Prehybridization Solution1 (in mixing order)
**Denature 10 minutes and place on ice before adding to mixture 1 Can mix everything except SSDNA and place in oven to warm to 65oC prior to use.
Hybridization Solution1 (in mixing order)
**Denature 10 minutes and place on ice before adding to mixture 1 Can mix everything except SSDNA and place in oven to warm to 65oC prior to use.
Post Hybridization Washes Low Stringency Wash: 2X SSC, 0.5% SDS
High Stringency Wash: 0.1X SSC, 0.1% SDS
(based on method from T. Helentjaris, NPI) Safety: This protocol involves radiation. The same safety requirements for the oligolabeling protocol apply here.
1.
Air dry and store in seal-a-meal bags. NOTES: *The object in removing counts is to expose the blots to 0.1 N NaOH for just long enough to remove the 32P-labeled probe. This may vary from lab to lab and should be determined experimentally by re-exposing stripped blots to X-ray film.
Re-Use Washes
High Stringency Wash: 0.1X SSC, 0.1% SDS
Reconditioning Solution: 0.2 M Tris-7.5, 0.1X SSC, 0.2% SDS
Making Glycerol Stocks from Stabs
Safety: Sterile technique is requisite to maintain stock quality and viability. Potential allergic reactions to the selective agents should be taken into consideration. Mishandling of the selective agents can cultivate a hypersensitivity. Properly dispose of unwanted materials that have come in contact with a bacterial culture. Note: All work should be performed in sterile conditions. 1. Streak the bacteria onto a plate containing the appropriate selective agent (ex. Ampicillin) using a sterile toothpick or transfer loop. The plate should be made to allow isolation of single colonies. We typically use the three-phase streaking technique diagrammed below. Be sure to use a new sterile toothpick or resterilize the transfer loop between streak number 1 and 2 and between streak number 2 and 3.
2. Grow the plate overnight at 37oC. 3. Using a sterile toothpick or pipet tip, touch a single colony and then drop the toothpick/tip into 4 ml of LB liquid media with the appropriate selective agent. 4. Grow this culture overnight at 37oC. 5. Transfer 700 µl of the liquid culture into a cryovial containing 300 µl of sterile glycerol. Cap the cryovial and mix well by inverting. Place this vial into a -80oC freezer for permanent storage. All future DNA isolations of the clone should be done from this stock. Use the rest of the culture to prepare Plasmid DNA following the plasmid DNA Miniprep procedure.
To grow bacteria from the glycerol stock for DNA isolation: 1. Remove the glycerol stock from the freezer on ice. DO NOT THAW THE GLYCEROL STOCK. This reduces viability. Open the glycerol vial under sterile conditions. Using a sterile toothpick or pipet tip scrap a small amount of the frozen glycerol stock out and drop it into liquid culture media on the toothpick/tip. Return the stock to the -80oC freezer as soon as possible. 2. Grow culture overnight at 37oC and follow the plasmid DNA Miniprep procedure.
(based on method of Birnboim, 1983, Methods of Enzymol. 100:243-255) Safety: Work with phenol and chloroform should be carried out in a fume hood. Lab coat, safety glasses and latex gloves should be worn. Used phenol and chloroform extraction material should be disposed of properly. Properly dispose of unwanted materials that have come in contact with a bacterial culture.
Solution I: 25 mM Tris pH 8.0, 10 mM EDTA, 50 mM glucose
Solution I may be prepared as a 10X stock solution and stored -20.C, in small aliquots. For use: thaw, dilute, and add lysozyme.
Solution II: 0.2 M NaOH, 1.0% SDS
Solution III: 3 M KOAc, pH 5.5 Dissolve 29.5 g potassium acetate in 60 ml dH2O. Add enough glacial acetic acid to bring pH to 5.5 (approx. 11 ml). Bring final volume to 100 ml.
Plasmid Mini-preps using the Wizard DNA Purification Kit We also use the Wizard Miniprep DNA Purification System from Promega successfully. 1. Grow 4 ml culture overnight with selection. 2. Use the Wizard Kit according to manufacturers instructions. Be sure to use 65oC TE pH 8.0 to release DNA from spin columns in final step. YIELD: 10 ug DNA from 4 ml culture.
Digestion/Isolation of Plasmid Inserts
3. Stop reaction by adding 5 µl of 5X SGB (Sample Gel Buffer) .
4. Prepare a 1.0% LMP agarose gel. Heat the agar a little slower than regular agarose to minimize foaming. Cool agarose to 55.C and pour gel. Once the gel has set, place at 4.C to cool. (BRL LMP Agarose (#5517UA) appears to be a little easier to work with than other LMP agaroses.) 5. Remove the gel from the refrigerator. Place into gel apparatus, load the samples, and run until the dye has migrated at least 7 cm. 6. Stain the gel in 1 µg/ml ethidium bromide (50 µl of 10 mg/ml ethidium bromide in 500 ml dH2O) for 20 min. Destain in dH2O for 20 min. 7. When visualizing the bands, it is best to minimize exposure to UV by either using a hand-held longwave UV lamp or by leaving the gel on a UV transparent tray and placing on a transilluminator. 8. Photograph the gel. Determine whether insert sizes are correct. If so, cut each out with a scalpel and place into a Sarstedt tube (#72-694-006). 9. Dilute each sample in TE pH 8.0 to the desired concentration (10 ng/ µl). We approximate the final volume from the intensity of the band in the gel. 10. Mix the agar-TE mixture by heating at 65-70.C for 5-10 min. Store at 4.C in tightly sealed tubes. Inserts are stable for oligolabeling for several years. qX174 / l HindIII Molecular Weight Marker 15 ul qX174 (BRL #15611-015) 15 ul l HindIII (BRL # 15612-013) 50 ul 5X SGB (Sample Gel Buffer) 420 ul dH2O Load 50 ul in gel to size inserts with. Store at 4.C. (Suzanne Livingston, University of Minnesota)
Note: If MgCl2 is not in your 10X buffer, add 6 µl of 25mM MgCl2 per reaction for a reaction concentration of 1.5mM and add 6 µl less H20.
Beckmann DU-65 Spectrophotometer The following are instructions for a program written for a Beckmann DU-65 Spectrophotometer. The program is designed to enable the user to quickly take 260nm and 280nm readings of many samples and from these calculate 260/280 ratios, concentrations, total DNA, and the amount of TE needed to bring the samples to a specified concentration. 1. Turn on U.V. Light source for spectrophotometer. It takes approximately 1 minute for the U.V. light to come on, it is however best to wait 15 minutes for the lamp to warm. When the light is on it will be indicated by the U.V. letters in the LCD display changing from lower case to upper case. Make sure that the printer is powered and on-line. 2. Press the program button. This will display programs available to the user. Select Program 0: DNA by pressing either STEP or BSTP. 3. When Program 0: DNA is displayed in the LCD display, press R/S. 4. You will be cued for the following information: a. STORED INFO Y 1 N 0 Are you recalculating values for previously stored information? Press 1 then ENTER if Yes or 0 then ENTER if No. b. DILUTION? What dilution factor is your sample aliquot? The default is 1:50. If your sample is diluted to something other than 1:50 press that number and press ENTER. RFLP Lab uses 150 (5 µl sample in 745 µl TE). c. RNA FACTOR? The default RNA factor is 1. This indicates the fact that RNase has been used on the sample and the sample contains no RNA. Otherwise a RNase factor of 5 is generally used with Maize. Press the number and then ENTER. To enter the default simply press ENTER. d. RESUS. VOLUME? At what volume is your final sample from which this aliquot has been taken? The default value is 1500 µl. e. FINAL µg/µl? At what concentration would you like your sample, from which this aliquot has been taken, to be diluted too? The default is 0.2 µg/µl. 5. You will be asked to insert a blank. The blank is whatever liquid you have used to dilute your sample aliquot. This will be used to calibrate the instrument. Press R/S. This is very important since all future calculations are dependant upon it. 6. You will then be asked to insert each sample. Press R/S and the spectrophotometer will sip the sample, calculate concentrations, and request the next sample. This will continue indefinitely until PROG is pressed. 7. Once all of your samples have been checked, values for resuspension and so forth can be recalculated. This is done by rerunning the PROG O: DNA. When prompted at the beginning of the program about STORED INFO Y 1 N 0, enter a 1 for Yes. You will then be prompted as previously for information, however instead of taking samples the spectrophotometer will recalculate values from figures stored from the last run of samples. These are stored in the scanning buffer. PROGRAM LISTING: PROG O:DNA 000: Strt 001: disp 5 002: ABS 003: 1.0 004: STO 006 005: MSG cSTO 006: MSG RED 007: MSG INFO 008: MSG Y 1 009: MSG NO 010: CALL ENTR 011: STO 008 012: 50. 013: STO 000 014: MSG DILU 015: MSG TION 016: MSG ? 017: CALL COUT 018: CALL ENTR 019: STO 000 020: 4.0 021: CALL BLNK 022: RCL 000 023: CALL FOUT 024: CALL CRLF 025: 1.0 026: STO 001 027: MSG RNA 028: MSG FACT 029: MSG OR? 030: CALL COUT 031: CALL ENTR 032: STO 001 033: 5.0 034: CALL BLNK 035: RCL 001 036: CALL FOUT 037: CALL CRLF 038: 1.5000E+03 039: STO 002 040: MSG RESU 041: MSG S VO 042: MSG L? 043: CALL COUT 044: CALL ENTR 045: STO 002 046: 2.0 047: CALL BLNK 048: RCL 002 049: CALL FOUT 050: CALL CRLF 051: 2.000E-01 052: STO 003 053: MSG FINA 054: MSG L uG 055: MSG uL? 056: CALL COUT 057: CALL ENTR 058: STO 003 059: 8.0 060: CALL BLNK 061: RCL 003 062: CALL FOUT 063: CALL CRLF 064: CALL CRLF 065: 1.0 066: RCL 008 067: x=y 068: GOTO READ 069: 1.0 070: CALL CHAN 071: LBL READ 072: MSG INS 073: MSG ERT 074: MSG BLAN 075: MSG K 076: R/S 077: CALL FILL 078: 2.8000E+02 079: LMDA 080: CALB 081: 2.6000E+02 082: LMDA 083: CALB 084: 1.0 085: CALL CHAN 086: rtn PROG l:HEADER 000: Strt 001: 57. 002: CALL BLNK 003: MSG cTOT 004: MSG AL 005: CALL COUT 006: 9.0 007: CALL BLNK 008: MSG cTE 009: CALL COUT 010: CALL CRLF 011: 35. 012: CALL ASCI 013: 5.0 014: CALL BLNK 015: MSG cSAM 016: MSG PLE 017: CALL COUT 018: 1.0 019: CALL BLNK 020: 4.0 021: CALL BLNK 022: MSG cA26 023: MSG 0 024: CALL COUT 025: 5.0 026: CALL BLNK 027: MSG cA28 028: MSG 0 029: CALL COUT 030: 5.0 031: CALL BLNK 032: MSG c260 033: CALL COUT 034: 47. 035: CALL ASCI 036: MSG c280 037: CALL COUT 038: 5.0 039: CALL BLNK 040: MSG cuG 041: CALL COUT 042: 47.0 043: CALL ASCI 044: MSG cuL 045: CALL COUT 046: 5 . 0 047: CALL BLNK 048: MSG cuG 049: MSG DNA 050: CALL COUT 051: 5.0 052: CALL BLNK 053: MSG cTO 054: MSG ADD 055: CALL COUT 056: CALL CRLF 057: CALL LINE 058: CALL CRLF 059: 2.0 060: CALL CHAN 061: rtn PROG 2:LOOP 000: Strt 001: 1.0 002: RCL 008 003: x=y 004: GOTO LOOP 005: 3.0 006: CALL CHAN 007: lbl LOOP 008: disp 3 009: RCL 006 010: STO 012 011: MSG IN 012: MSG RT 013: MSG AMPL 014: MSG E 015: R/S 016: CALL FILL 017: 2.6000E+02 018: LMDA 019: READ 020: STO 004 021: RCL 006 022: 2.0 023: * 024: STO 009 025: RCL 004 026: CALL STOR 027: 2.8000E+02 028: LMDA 029: READ 030: STO 005 031: RCL 009 032: 1.0 033: + 034: RGL 005 035: CALL STOR 036: RCL 006 037: CALL FOUT 038: 1.0 039: + 040: STO 006 041: disp 5 042: 2.0 043: CALL BLNK 044: 10. 045: STO 010 046: lbl LINE 047: 95. 048: CALL ASCI 049: dec 010 050: GOTO LINE 051: 2.0 052: CALL BLNK 053: RCL 004 054: CALL FOUT 055: 4.0 056: CALL BLNK 057: RCL 005 058: CALL FOUT 059: 4.0 060: CALL BLNK 061: RCL 004 062: RCL 005 063: / 064: CALL FOUT 065: 7.0 066: CALL BLNK 067: 5.000 068: RCL 001 069: / 070: RCL 004 071: * 072: RCL 000 073: * 074: STO 007 075: CALL FOUT 076: 5.0 077: CALL BLNK 078: disp 6 079: RCL 007 080: RCL 002 081: * 082: CALL FOUT 083: 5.0 084: CALL BLNK 085: RCL 002 086: RCL 007 087: * 088: RCL 003 089: / 090: RCL 002 091: - 092: CALL FOUT 093: CALL CRLF 094: GOTO LOOP 095: rtn PROG 3: REPEAT 000: Strt 001: lbl READ 002: disp 3 003: RCL 006 004: CALL FOUT 005: disp 5 006: 2.0 007: CALL BLNK 008: 10. 009: STO 010 010: lbl LINE 011: 95. 012: CALL ASCI 013: dec 010 014: GOTO LINE 015: 2.0 016: CALL BLNK 017: RCL 006 018: 2.0 019: * 020: STO 009 021: CALL LOAD 022: STO 004 023: CALL FOUT 024: 4.0 025: CALL BLNK 026: RCL 009 027: 1.0 028: + 029: CALL LOAD 030: STO 005 031: CALL FOUT 032: 4.0 033: CALL BLNK 034: RCL 004 035: RCL 005 036: / 037: CALL FOUT 038: 7.0 039: CALL BLNK 040: 5.0000E-02 041: RCL 001 042: / 043: RCL 004 044: * 045: RCL 000 046: * 047: STO 007 048: CALL FOUT 049: 5.0 050: CALL BLNK 051: disp 6 052: RCL 007 053: RCL 002 054: * 055: CALL FOUT 056: 5.0 057: CALL BLNK 058: RCL 002 059: RCL 007 060: * 061: RCL 003 062: / 063: RCL 002 064: - 065: CALL FOUT 066: RCL 006 067: 1.0 068: + 069: STO 006 070: CALL CRLF 071: RCL 006 072: RCL 012 073: X<=Y 074: GOTO REPE 075: rtn Dissolve 2.0 g Ficoll 400, 2.0 g polyvinylpyrrolidone (40,000 MW), and 2.0 g BSA (Fraction V, Sigma) in sterile dH20 to a final volume of 100 ml. Vacuum filter. Store at -20.C in 10 ml aliquots.
Dissolve 50 g dextran sulphate (Pharmacia #17-0340-02) in sterile dH20 to a final volume of 100 ml. Solution is extremely viscous and will take a long time to dissolve.
0.1 M DTT Dissolve 1.55 g dithiothreitol in 10 ml of 0.01 M NaOAC-5.2. Dilute 1:10 with 0.01 M NaOAC-5.2. Sterilize by filtration. Store in 100 µl aliquots at -20.C.
Dissolve 186.12 g Na2EDTAï2H20 (Disodium Ethylenediamine Tetraacetate) in approx. 750 ml of dH2O containing 20 gm of NaOH pellets (20 gm in 1 liter for 1 liter of 0.5 M NaOH). Check pH. If necessary, bring pH to 8.0 with additional NaOH pellets. After EDTA is in solution, bring to 1000 ml with dH2O. Autoclave.
Dissolve 100 mg of ethidium bromide in 10 ml dH2O . Wrap tube in aluminum foil and store at 4.C. CAUTION: EtBr is extremely mutagenic.
1 M MgCl2 Dissolve 20.33 g MgCl2 ï 6H2O in dH2O to a final volume of 100 ml. Autoclave.
Dissolve 7.71 g ammonium acetate in dH2O to a final volume of 100 ml. Filter sterilize.
Dissolve 57.83 g ammonium acetate in dH2O to a final volume of 100 ml. Filter sterilize.
Dissolve 292.2 g NaCl in dH2O to a final volume of 1000 ml. Autoclave.
Dissolve 20.5 g sodium acetate (anhydrous) in dH2O to a final volume of 100 ml. Autoclave.
Dissolve 40 g NaOH in dH2O to a final volume of 1000 ml. Autoclave. (Best to weigh approx. 40 g of pellets, then determine correct final volume for a 1 N solution.)
Phenol (equilibrated) Purchased from Amersco #945. Add included buffer and mix. Store at 4.C. Old way: Equilibrate melted Ultra-Pure phenol (BRL Cat# 5509UA, redistilled nucleic acid grade) by adding an equal volume of dH2O. Shake well and allow to separate (phenol equilibrated with water can be aliquoted into 50 ml tubes and stored at -20.C until needed), vacuum aspirate off aqueous (top) layer. Repeat equilibration two more times with 1 M Tris-HCl pH 8.0, and once with TE pH 8.0. Leave a small layer of TE on the phenol. Aliquot equilibrated phenol into 50 ml tubes with caps, wrap each in foil, and store at 4.C.
1 M NaH2PO4 Dissolve 138 g of sodium phosphate, monobasic, monohydrate in dH2O to a final volume of 1000 ml. Autoclave.
1 M Na2HPO4
0.75 M NaPO4 pH 3.5: PEI Cellulose TLC Buffer Add 1 M H3P04 to 75 ml of 1 M NaH2P04 to bring pH to 3.5 with phosphoric acid. Dilute with dH2O to a final volume of 100 ml.
1 M NaPO4 pH 6.5: Blot Transfer Buffer For approximately 1 liter, start with 660 ml 1 M NaH2P04 and add 1 M Na2HP04 to bring pH to 6.5 (approx. 330 ml). -OR -
Adjust pH to 6.5 with NaOH pellets.
1 M H3PO4 Add 5.5 ml of phosphoric acid (85%) to dH2O to a final volume of 100 ml. Autoclave.
Dissolve 100 mg of RNAse (Sigma #R4875) in 10 mM Tris pH 7.5, 15 mM NaCl. Heat in boiling water for 15 min and allow to cool slowly to room temperature. Dispense into aliquots and store at -20.C, working stock may be stored at 4.C.
Dilute RNAse T1 (Sigma #R8251) with 10 mM Tris pH 7.5, 15 mM NaCl to 500 U/ml. Heat in boiling water for 15 min and allow to cool slowly to room temperature. Aliquot and store at -20.C.
10 mg/ml Salmon Sperm DNA: SS DNA Dissolve 100 mg salmon sperm DNA (Sigma #D1626) in TE - 8.0 to a final volume of 10 ml by rotating overnight. Place in sonicator for 48 hour or autoclave. Shear the DNA by passing through a 18 gauge needle 3-4 times. Repeat with a 22 or 25 gauge needle. Aliquot in 15ml tubes. Boil in water for 10 min. Store at -20.C. Thaw and boil in water for 10 min. prior to use. Dissolve 200 g sodium dodecyl sulfate in dH20 to final volume of 1000 ml. Can use a low grade (Sigma #L5750) for hyb washes, etc. and a better grade (Sigma #L4390) for hyb solution, plasmid preps, stop solutions, etc.
25X SSC (3.7 M NaCl, 0.375 M Citric Acid, Trisodium salt dihydrate)
Adjust pH to 7.4 with 1 N HCl.
Dissolve 255 mg spermidine (Sigma #S2501) in dH2O to a final volume of 10 ml. Filter sterilize. Store at -20.C, working stock may be stored at 4.C.
1 M Tris pH 7.5, pH 8.0 Dissolve 121 g Tris-Base in approx. 750 ml dH2O . Add conc. HCl until desired pH is reached (75 ml HCl = pH 7.5, 49 ml HCl = pH 8.0). Bring solution to 1000 ml with dH2O. Autoclave.
TE pH 8.0 ( 10 mM Tris pH 8.0, l mM EDTA pH 8.0)
Autoclave.
Per liter:
Adjust pH to 7.5 with 1 M NaOH. LB + Amp* Autoclave and let cool to less than 60oC. Add 6 mls ampicillin per liter of sterile LB media. Do not autoclave solution containing antibiotics. LB + Amp* for Plates Add 15 g Bacto-agar per liter of LB media. Dissolve agar in microwave, autoclave and let cool to less than 60oC. Add 6 mls ampicillin per liter of sterile LB media, pour 25 ml per plate. LB + Amp* for Stabs Add 7 g Bacto-agar per liter of LB media. Dissolve agar in microwave, autoclave. Let cool to to less than 60oC. Add 6 mls ampicillin per liter of sterile LB media, pipet stabs. *or other selective agent.
TB Media Per liter:
Mix well and sterilize by autoclaving for 20 min at 15 lb/sq. in. Allow the solution to cool to 60oC, add 100 ml of a sterile solution of 0.17 M KH2PO4, 0.72 M K2HPO4 (dissolve 2.31 g of KH2PO4 and 12.54 g of K2HPO4 in 90 ml of dH20. Adjust the volume to 100 ml after salts have dissolved and sterilize by autoclaving for 20 min at 15 lb/sq. in.).
Ampicillin (25 mg/ml) Add 12.5 g Ampicillin, (sodium salt), Sigma #A9518, to 500 ml sterile dH20. Mix. Aliquot into 15 ml tubes and store at -20oC. Use 6 mls in 1 liter of media.
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