RFLP
Protocols
Table of Contents
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 -60ƒC 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 <-60ƒC. Samples should be dried in 72 hours. Typically, fresh weight approximately 10 x dry weight.
Leaf samples may alternatively be stored at -20oC or -80ƒC until
lyophilization.
5. Dried leaf samples may be stored in sealed plastic bags at room temperature for short periods of time (<1 month) or at -20ƒC 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 65ƒC 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 65ƒC 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 4ƒC.
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).
|
STOCK |
50 ml |
100 ml |
250 ml |
400 ml |
500 ml |
600 ml |
|
dH2O |
36.5 ml |
73 ml |
292 ml |
365 ml |
438 ml |
182.5 ml |
|
1 M Tris 7.5 |
5 ml |
10 ml |
25 ml |
40 ml |
50 ml |
60 ml |
|
7 ml |
14 ml |
35 ml |
56 ml |
70 ml |
84 ml |
|
|
1 ml |
2 ml |
5 ml |
8 ml |
10 ml |
12 ml |
|
|
14 M BME2 |
0.5 ml |
1 ml |
2.5 ml |
4 ml |
5 ml |
6 ml |
|
CTAB3 |
0.5 g |
1 g |
2.5 g |
4 g |
5 g |
6 g |
1
Make fresh before use.2
Add BME (ß-mercaptoethanol) to warmed buffer (60-65ƒC), just prior to use.3
CTAB = Mixed alkyltrimethyl-ammonium bromide (Sigma).
WASH 1: 76% EtOH, 0.2 M NaOAc
|
STOCK |
|
100 ml |
200 ml |
300 ml |
400 ml |
500 ml |
|
Absolute EtOH |
|
76 ml |
152 ml |
228 ml |
304 ml |
380 ml |
|
|
8 ml |
16 ml |
24 ml |
32 ml |
40 ml |
|
|
dH2O |
|
16 ml |
32 ml |
48 ml |
64 ml |
80 ml |
WASH 2: 76% EtOH, 10 mM NH4OAc
|
STOCK |
100 ml |
200 ml |
300 ml |
400 ml |
500 ml |
|
Absolute EtOH |
76 ml |
152 ml |
228 ml |
304 ml |
380 ml |
|
1 ml |
2 ml |
3 ml |
4 ml |
5 ml |
|
|
dH2O |
23 ml |
46 ml |
69 ml |
92 ml |
115 ml |
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 4ƒC for up to 6 months. Store at -20ƒC 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)
|
STOCK |
[FINAL] |
|
|
10X Buffer* |
1X |
30 µl |
|
2.5 µM |
7.5 µl |
|
|
dH2O* |
|
to bring rxn volume to 300 µl |
|
Enzyme (U=units)* |
2.5 U/m g DNA |
Dependent on amount of DNA |
|
DNA |
|
|
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 -80ƒC 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)
|
STOCK |
10 ml |
50 ml |
100 ml |
500 ml |
1000 ml |
|
ddH2O |
4 ml |
20 ml |
40 ml |
200 ml |
400 ml |
|
1 M Tris pH 8.0 |
0.5 ml |
2.5 ml |
5 ml |
25 ml |
50 ml |
|
Glycerol |
5 ml |
25 ml |
50 ml |
250 ml |
500 ml |
|
100 ul |
500 ul |
1 ml |
5 ml |
10 ml |
|
|
250 ul |
1.25 ml |
2.5 ml |
12.5 ml |
25 ml |
|
|
Bromphenol Blue* |
15 mg |
75 mg |
150 mg |
750 mg |
1.5 g |
|
Xylene Cyanole |
15 mg |
75 mg |
150 mg |
750 mg |
1.5 g |
*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 60ƒC.
|
Gel Size |
Agarose (0.7%) |
Sample Volume |
|
|
11 x 14 cm |
0.70 g |
100 ml |
|
|
11 x 20 |
1.05 g |
150 ml |
20 µl |
|
20 x 25 |
2.10 g |
300 ml |
20 µl |
|
20 x 25 |
2.45 g |
350 ml |
25 µl |
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.
|
STOCK |
1 liter |
5 liters |
10 liters |
15 liters |
20 liters |
|
Tris Base |
242 g |
1210 g |
2420 g |
3630 g |
4840 g |
|
Glacial Acetic Acid |
57.1 ml |
285.5 ml |
571 ml |
856.5 ml |
1142 ml |
|
100 ml |
500 ml |
1000 ml |
1500 ml |
2000 ml |
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 4ƒC.
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 60ƒC.
|
Gel Size |
Agarose (0.7%) |
Sample Volume |
|
|
11 x 14 cm |
1.40 g |
220 ml |
50 µl |
|
11 x 20 |
2.10 g |
330 ml (170 + 160)* |
50 µl |
|
20 x 25 |
4.20 g |
675 ml (350 + 325)* |
50 µl |
|
20 x 25 |
4.90 g |
700 ml (350 + 350) |
55-60 µl |
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 60ƒC 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.
|
Gel Size |
Blotting Paper Size |
Membrane Size |
|
11 x 14 cm |
11.5 x 14.5 cm (4 1/2" x 5 3/4") |
11 x 14 cm (4 1/4" x 5 1/2") |
|
11 x 20 cm |
11.5 x 20.5 cm (4 1/2" x 8") |
11 x 20 cm (4 1/4" x 7 3/4") |
|
20 x 25 cm |
20.5 x 25.5 cm (8" x 10") |
20 x 25 cm (7 3/4" x 9 3/4") |
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)
|
STOCK |
500 ml |
1 L |
2 L |
5 L |
10 L |
20 L |
|
NaOH |
8.00 g |
16.00 g |
32.00 g |
80.00 g |
160.00 g |
320.00 g |
|
NaCl |
8.76 g |
35.04 g |
70.08 g |
175.20 g |
350.40 g |
700.80 g |
Neutralizing Buffer: 0.5 M Tris-7.5, 1.5 M NaCl (1 liter/gel)
|
STOCK |
500 ml |
1 L |
2 L |
5 L |
10 L |
20 L |
|
Tris-HCl |
31.75 g |
63.50 g |
127.00 g |
317.50 g |
635.00 g |
1270.00 g |
|
Tris-Base |
5.90 g |
11.80 g |
23.60 g |
59.00 g |
118.00 g |
236.00 g |
|
NaCl |
43.80 g |
87.60 g |
175.20 g |
438.00 g |
876.00 g |
1752.00 g |
Adjust pH to 7.5 with conc. HCl
OR
|
STOCK |
500 ml |
1 L |
2 L |
5 L |
10 L |
20 L |
|
Tris-Base |
30.25 g |
60.50 g |
121.00 g |
302.50 g |
605.00 g |
1210.00 g |
|
Conc. HCl |
12.5 ml |
25.0 ml |
50.0 ml |
125.0 ml |
250.0 ml |
500.0 ml |
|
NaCl |
43.80 g |
87.60 g |
175.20 g |
438.00 g |
876.00 g |
1752.00 g |
Transfer Buffer: 25 mM NaPO4, pH 6.5 (4 liters/gel)
|
STOCK |
1 L |
2 L |
5 L |
10 L |
20 L |
|
1 M NaPO4 pH 6.5 |
25 ml |
50 ml |
125 ml |
250 ml |
500 ml |
|
STOCK |
250 ml |
500 ml |
750 ml |
1000 ml |
1500 ml |
2000 ml |
|
20 ml |
40 ml |
60 ml |
80 ml |
120 ml |
160 ml |
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:
|
STOCK |
Full RXN |
Half RXN |
|
||||
|
OLB (50 mg/ul) |
1X |
10 ul |
5 ul |
|
|||
|
BSA (1 mg/ml) |
5 ug |
5 ul |
2.5 ul |
|
|||
|
50 uCi |
5 ul |
2.5 ul |
|
||||
|
Klenow (500 U/84 ul) |
2.5 U |
0.5 ul |
0.25 ul |
|
|||
|
DNA (5 ng/ul) |
50 ng |
10 ul |
5 ul |
|
|||
|
ddH2O |
|
20 ul |
10 ul |
|
|||
Volume Totals: |
|
50.5 ul |
25.25 ul |
|
|||
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 95ƒC 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 95ƒC 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 -20ƒC. 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 -20ƒC. 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 -20ƒC. 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 -20ƒC 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 -20ƒC. 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 -20ƒC. 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 -20ƒC. 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 -20ƒC. Mark tubes with green and black tops.
OLB Stop Mix
|
STOCK |
50 ml |
100 ml |
200 ml |
|
1 M Tris -HCl, 7.5 |
l.0 ml |
2.0 ml |
4.0 ml |
|
0.2 ml |
0.4 ml |
0.8 ml |
|
|
0.2 ml |
0.4 ml |
0.8 ml |
|
|
6.25 µl |
12.5 µl |
25.0 µl |
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 4ƒC.
Removal of
Unincorporated dNTP's
by Spin Columns
(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 95ƒC 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.
(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 65ƒC, 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.
|
# Washes |
Time/Wash |
Temp/Wash |
Solution |
|
3 |
5-10 minutes |
Room temp. |
|
|
1 |
30 minutes |
65oC |
|
|
2 |
45 minutes |
65oC |
Prehybridization Solution1 (in mixing order)
|
Stock |
100 ml |
250 ml |
500 ml |
1000 ml |
2000 ml |
|
dH20 |
70 ml |
175 ml |
350 ml |
700 ml |
1400 ml |
|
1 M Tris pH 8.0 |
5 ml |
12.5 ml |
25 ml |
50 ml |
100 ml |
|
2.0 ml |
5.0 ml |
10 ml |
20 ml |
40 ml |
|
|
20 ml |
50 ml |
100 ml |
200 ml |
400 ml |
|
|
1.0 ml |
2.5 ml |
5 ml |
10 ml |
20 ml |
|
|
1.0 ml |
2.5 ml |
5 ml |
10 ml |
20 ml |
|
|
1.0 ml |
2.5 ml |
5 ml |
10 ml |
20 ml |
**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)
|
Stock |
100 ml |
250 ml |
500 ml |
1000 ml |
2000 ml |
|
dH20 |
50 ml |
125 ml |
250 ml |
500 ml |
1000 ml |
|
20 ml |
50 ml |
100 ml |
200 ml |
400 ml |
|
|
1 M Tris pH 8.0 |
5 ml |
12.5 ml |
25 ml |
50 ml |
100 ml |
|
2.0 ml |
5.0 ml |
10 ml |
20 ml |
40 ml |
|
|
20 ml |
50 ml |
100 ml |
200 ml |
400 ml |
|
|
1.0 ml |
2.5 ml |
5 ml |
10 ml |
20 ml |
|
|
1.0 ml |
2.5 ml |
5 ml |
10 ml |
20 ml |
|
|
1.0 ml |
2.5 ml |
5 ml |
10 ml |
20 ml |
**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
|
STOCK |
500 ml |
1 L |
2 L |
5 L |
10 L |
20 L |
|
40 ml |
80 ml |
160 ml |
400 ml |
800 ml |
1600 ml |
|
|
12.5 ml |
25 ml |
50 ml |
125 ml |
250 ml |
500 ml |
High Stringency Wash: 0.1X SSC, 0.1% SDS
|
STOCK |
500 ml |
1 L |
2 L |
5 L |
10 L |
20 L |
|
2 ml |
4 ml |
8 ml |
20 ml |
40 ml |
80 ml |
|
|
2.5 ml |
5 ml |
10 ml |
25 ml |
50 ml |
100 ml |
(based on method from T. Helentjaris, NPI)
Safety: This protocol involves radiation. The same safety requirements for the oligolabeling protocol apply here.
1.
|
# Washes |
Time/Wash |
Temp/Wash |
Solution |
Amount |
|
1 |
10 minute |
Room Temp. |
3 L / 20 blots |
|
|
1 |
4 minute* |
Room Temp. |
3 L / 20 blots |
|
|
1 |
15 minute |
Room Temp. |
3 L / 20 blots |
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