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Project Documentation & Protocols: Maize Mapping Project: RFLP Protocols

Foreword

Lyophilization

Grinding

Genomic DNA Isolation

UV Quantification of DNA

Restriction Digests of Genomic DNA

Neutral Agarose Gel Electrophoresis

Double Thick Gels

Southern Blotting

Oligolabeling

Removal of Unincorporated dNTP's
by Spin Columns

Checking Incorporation by PEI Cellulose TLC

Prehybridizations and Hybridizations

Stripping Blots for Re-Use

Making Glycerol Stocks from Stabs

Plasmid Mini-Preps

Digestion/Isolation of Plasmid Inserts

PCR Protocol For Plasmids

Beckmann DU-65 Spectrophotometer
DNA Quantifation Program

Stock Solutions

 

 

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.

 

Foreword

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,

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).

Lyophilization

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.


Leaf samples may alternatively be stored at -20oC or -80.C 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 -20.C for several years. Desiccant should be included in the plastic bag to prevent the samples from taking on water.

Grinding

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. Grind to a fine powder with a mechanical mill1, into a plastic scintillation vial or any other appropriate plastic container that can be closed air tight. If the plant weighed less than 4 g (fresh weight), grind to a powder with a mortar and pestle in the presence of a pinch of acid washed sand. The finer the grind, the greater the yield of DNA from a given amount of material.

     

     

  2. Store ground samples tightly capped at -20.C. Samples are stable for several years. For larger volumes of ground tissue, it is important to place a desiccant pack in each sample bottle to prevent water accumulation. Desiccant packs may be purchased from Sigma Chemical (#S-8394).

     

1 Cyclone sample mill

Genomic DNA Isolation

(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 l 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 l 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 l 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.

1Note 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).

 

CTAB Extraction Buffer1

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

5 M NaCl

7 ml

14 ml

35 ml

56 ml

70 ml

84 ml

0.5 M EDTA pH 8.0

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 (-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

STOCK

 

100 ml

200 ml

300 ml

400 ml

500 ml

Absolute EtOH

 

76 ml

152 ml

228 ml

304 ml

380 ml

2.5 M NaOAc

 

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 M NH4OAc

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 l of each DNA sample to 745 l TE (dilution factor, d.f., = 150), read OD260 and OD280 to determine purity. Dilute samples to 0.4 g/l (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 (g/ l) = [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.

.

Restriction Digests of

Genomic DNA

(based on method from T. Helentjaris, NPI)

STOCK

[FINAL]

Per 300 l RXN

10X Buffer*

1X

30 l

0.1 M Spermidine*

2.5 M

7.5 l

dH2O*

 

to bring rxn volume to 300 l

Enzyme (U=units)*

2.5 U/m g DNA

Dependent on amount of DNA

DNA

 

 

 

 

  1. Determine g of sample DNA to be digested and then the volume of sample DNA necessary. Aliquot DNA into 1.5 l microfuge tubes.
  2. Determine the units (U) of enzyme necessary to digest DNA sample. In general, it is best to use 2.5 U/g DNA to prevent partial digestions.
  3. FOR LARGE NUMBERS OF DIGESTS WITH THE SAME ENZYME: Determine the total number of reactions, i.e. the sum of the different DNA samples to be digested by the same enzyme. Add 2-3 reactions to allow for pipetting errors. Multiply this number by the volumes in the per 300 l RXN column above for all components with an asterisk.
  4. Prepare bulk mixture on ice of all components with an asterisk, adding enzyme last.
  5. Aliquot bulk mix into reaction tubes to a final volume of 300 l each. Mix well (do not vortex).
  6. Incubate at 37.C for time recommended by manufacturer.
  7. Stop the reaction by heating at 65oC for 15-20 minutes.

REPRECIPITATION OF DIGESTED DNA:

1. Add 20 l of 5 M NaCl to each 300 l rxn.

2a. Add 750 l (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 l 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 l of TE pH 8.0 and 5 l 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

0.5 M EDTA pH 8.0

100 ul

500 ul

1 ml

5 ml

10 ml

20% SDS

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 60.C.

 

Gel Size

Agarose (0.7%)

1X TAE Gel Buffer

Sample Volume

11 x 14 cm

0.70 g

100 ml

20 l

11 x 20

1.05 g

150 ml

20 l

20 x 25

2.10 g

300 ml

20 l

20 x 25

2.45 g

350 ml

25 l

 

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 g/ml ethidium bromide (50 l 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.

 

50X TAE Gel Buffer

 

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

0.5 M EDTA pH 8.0

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 4.C.

 

Double Thick Gels

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.

 

Gel Size

Agarose (0.7%)

1X TAE Gel Buffer

Sample Volume

11 x 14 cm

1.40 g

220 ml

50 l

11 x 20

2.10 g

330 ml (170 + 160)*

50 l

20 x 25

4.20 g

675 ml (350 + 325)*

50 l

20 x 25

4.90 g

700 ml (350 + 350)

55-60 l

 

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 l 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 g/ml ethidium bromide (50 l 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.

Southern Blotting

(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.

  1. Briefly, dip 2 sheets S & S precut gel blotting paper (20 x 25 cm sheets, #34550, GB002, Midwest Scientific) transfer buffer and place on top of membrane.
  2. Place a piece of x-ray film around each side of blot covering any blotting paper.
  3. Carefully, place a 2" stack of paper towels on top of the blotting paper. An optional light weight can be placed on top, if used with a flat surface to provide even pressure to blotting surface.

Note: Paper towels should cover entire area of gel and not extend beyond edge of membrane. This will short-circuit the transfer.

  1. Add transfer buffer to tray, so that the buffer level remains high during blotting process.
  2. Allow to transfer overnight (6-18 hours).
  3. Remove membrane and immediately place in 2X SSC. With gloved hand, briskly rub off any agar particles. Wash blot for 15 min., shaking in 2X SSC.
  4. Place damp filters (no standing water) in a UV Stratalinker and cross-link using 200 m j of energy. If using other membrane, cross-link according to the manufacturer specifications for that particular membrane.
  5. Place filters between clean Whatman filter paper. Bake 1 hour at 80oC for MSI Magnacharge membranes. If using other membrane, bake according to the manufacturer specifications. If blots are not going to be UV Stratalinked, then they must be baked.
  6. Briefly check transfer under UV light. If membrane was not previously labeled, label with Pigma pen or other suitable pen on DNA bound side.
  7. If blots are not going to be used for a week or more, store in a plastic bag in a cool, dry place (can be stored at 4.C).

 

 

 

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

 

 

2X SSC

 

STOCK

250 ml

500 ml

750 ml

1000 ml

1500 ml

2000 ml

25X SSC

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 l 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

 

dCT32P (3000 Ci/mM)

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 l of 5 ng/l per FULL RXN) and ddH2O to 500 l 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 l OLB reaction mix to 15 l 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 l 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 l of 1 M Tris -HCl and 40 l 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 l of OLB TE (dissolve directly in original bottle). Store in 30 l 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 l of OLB TE (dissolve directly in original bottle). Store in 30 l 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 l of OLB TE (dissolve directly in original bottle). Store in 30 l 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 gMgCl2.6H2O in 75 ml ddH2O. Bring pH to 8.0 and bring volume to 100 ml.

 

Solution A

To 1.0 ml Tris-MgCl Solution , add 18 l 2-mercaptoethanol, 5 l 100 mM dATP , 5 l 100 mM dGTP , and 5 l 100 mM dTTP . Store at -20.C for no more than 90 days. Mark tube with red and blue tops.

 

Solution B

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 l of OLB TE to the 50 Units of hexamers (Pharmacia 27-2166-01) in the shipping bottle, then add 738 l of Solution A, and 1850 l of Solution B. Store in 200 l aliquots at -20.C. Mark tubes with red and black tops.

- OR -

To make smaller quantities of OLB:

Mix 50 l Solution A, 125 l Solution B, and 75 l Hexamer Solution. Store in

100 l aliquots at -20.C. Mark tubes with red and black tops.

Hexamer Solution:

Dissolve 50 units of hexamers (Pharmacia 27-2166-01) in 1110 l of OLB TE . Store in 75 l aliquots at -20.C. 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

5 M NaCl

0.2 ml

0.4 ml

0.8 ml

0.5 M EDTA pH 8.0

0.2 ml

0.4 ml

0.8 ml

20% SDS

6.25 l

12.5 l

25.0 l

 

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.

 

Klenow

Purchase from BRL (# 18012-096).

 

dCT32P (3000 Ci/mM)

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
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 l 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 l of volume without touching column.

6. Add probe (95 l) to column and wash tube with additional 100 l 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 l 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.

  1. Place against film for 3 min. This exposure should be within the linear range of the film. Incorporated dCTP will stay at the origin, dCTP will be 2-3 cm above the origin and inorganic 32P will be at the front.

 

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.

  1. Denature probe by heating 7 min at 95.C and placing on ice until added to hybridization solution. Add the probe (25 ng DNA, 25 Ci dCT32P) and 8-10 ul of labeled standard per bag. Seal bag after removing large air bubbles. Hybridize overnight at 65.C, slowly rocking.
  2. Remove blot from bag and wash with good agitation as follows:

# Washes

Time/Wash

Temp/Wash

Solution

3

5-10 minutes

Room temp.

Low Stringency Wash

1

30 minutes

65oC

High Stringency Wash

2

45 minutes

65oC

High Stringency Wash

 

  1. Allow blots to just air dry (i.e., damp but no standing water nor dry spots) and wrap in plastic wrap or plastic sheet protectors (which are reusable, clean with ethanol prior to use).
  2. Expose blots to X-ray film at -80.C for appropriate time (overnight to 5 days). Use an intensifying screen on top of film.

 

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

0.5 M EDTA pH 8.0

2.0 ml

5.0 ml

10 ml

20 ml

40 ml

25X SSC

20 ml

50 ml

100 ml

200 ml

400 ml

100X Denhardt's

1.0 ml

2.5 ml

5 ml

10 ml

20 ml

20% SDS

1.0 ml

2.5 ml

5 ml

10 ml

20 ml

10 mg/ml Salmon Sperm DNA**

1.0 ml

2.5 ml

5 ml

10 ml

20 ml

 

**Denature 10 minutes and place on ice before adding to mixture

1Can 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

50% Dextran Sulfate

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

0.5 M EDTA pH 8.0

2.0 ml

5.0 ml

10 ml

20 ml

40 ml

25X SSC

20 ml

50 ml

100 ml

200 ml

400 ml

100X Denhardt's

1.0 ml

2.5 ml

5 ml

10 ml

20 ml

20% SDS

1.0 ml

2.5 ml

5 ml

10 ml

20 ml

10 mg/ml Salmon Sperm DNA**

1.0 ml

2.5 ml

5 ml

10 ml

20 ml

**Denature 10 minutes and place on ice before adding to mixture

1Can 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

25X SSC

40 ml

80 ml

160 ml

400 ml

800 ml

1600 ml

20% SDS

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

25X SSC

2 ml

4 ml

8 ml

20 ml

40 ml

80 ml

20% SDS

2.5 ml

5 ml

10 ml

25 ml

50 ml

100 ml

Stripping Blots for Re-Use

(based on method from T. Helentjaris, NPI)

Safety: This protocol involves radiation. The same safety requirements for the oligolabeling protocol apply here.

 

1. To remove counts and prepare for reuse, wash blots as follows:

# Washes

Time/Wash

Temp/Wash

Solution

Amount

1

10 minute

Room Temp.

High Stringency Wash

3 L / 20 blots

1

4 minute*

Room Temp.

32P-Stripping Solution

3 L / 20 blots

1

15 minute

Room Temp.

Reconditioning Solution

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. 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

STOCK

500 ml

1000 ml

1500 ml

2000 ml

2500 ml

3000 ml

25X SSC

2 ml

4 ml

6 ml

8 ml

10 ml

12 ml

20% SDS

2.5 ml

5 ml

7.5 ml

10 ml

12.5 ml

15 ml

32P-Stripping Solution: 0.1 N NaOH, 0.2% SDS

STOCK

500 ml

1000 ml

1500 ml

2000 ml

2500 ml

3000 ml

1 M NaOH

50 ml

100 ml

150 ml

200 ml

250 ml

300 ml

20% SDS

5 ml

10 ml

15 ml

20 ml

25 ml

30 ml

 

Reconditioning Solution: 0.2 M Tris-7.5, 0.1X SSC, 0.2% SDS

STOCK

500 ml

1000 ml

1500 ml

2000 ml

2500 ml

3000 ml

1 M Tris -7.5

100 ml

200 ml

300 ml

400 ml

500 ml

600 ml

25X SSC

2 ml

4 ml

6 ml

8 ml

10 ml

12 ml

20% SDS

5 ml

10 ml

15 ml

20 ml

25 ml

30 ml

Making Glycerol Stocks from Stabs

The UMC RFLP Laboratory routinely sent out clone requests as bacterial stabs. The following procedure should be followed on receipt of a clone in this form.

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 l of the liquid culture into a cryovial containing 300 l 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.

 

 

Plasmid Mini-Preps

(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.

  1. Grow 9 ml culture in LB media overnight at 37oC.
  2. Harvest cells by centrifuging entire culture in a 15 ml centrifuge tube for 10 min in a table-top centrifuge (1300-1500 x g). Discard supernatant.
  3. Resuspend cell pellet briefly by vortexing before adding in 200 l Solution I containing 5 mg/ml lysozyme (add lysozyme within 1 hour of use). Vortex and leave at room temperature for 5 min. It is easier to resuspend cells if they are vortexed before adding the lysozyme mix.
  4. Add 400 l Solution II, mix, and incubate 10 min on ice (solution should be clear).
  5. Add 300 l cold Solution III, mix, and incubate 15 min on ice.
  6. Centrifuge 15 min in table-top centrifuge, pour off supernatant into 1.5 ml microfuge tube.
  7. Add 600 l ice cold isopropanol, mix, centrifuge 5 min in microfuge (~12,000 rpm), drain and dry tube.
  1. Redissolve pellet in 185 l dH2O. Allow sample to resuspend overnight.
  2. Add 5 l of 10 mg/ml RNAse A and 5 l of 500 U/ml RNAse T1. Incubate at RT (or 37.C) for 15 min.
  3. Extract with 200 l phenol [or 200 l phenol/chloroform (1:1)].
  4. Centrifuge for 2 min in table-top centrifuge (1300-1500 x g). Transfer aqueous (upper) phase to new microfuge tube.
  5. Add 100 l 7.5 M NH4OAc.
  6. Add 750 l Absolute EtOH, mix, incubate at -80.C for 30 min, centrifuge 5 min at full-speed in microfuge.
  7. Wash pellet with 1 ml 75% EtOH, centrifuge 30 seconds in microfuge, pour off supernatant and dry tube in speed-vac (or vacuum desiccator).
  1. Dissolve pellet in 50 l TE pH 8.0 .
  2. Quantify plasmid DNA using protocol listed previously. Dilute sample with TE pH 8.0 to 1 g/ l. Store at -20.C. Sample should be usable for up to 6 months.

 

 

Solution I: 25 mM Tris pH 8.0, 10 mM EDTA, 50 mM glucose

STOCK

10 ml

20 ml

30 ml

40 ml

50 ml

1 M Tris -8.0

250 ml

500 ml

750 ml

1000 ml

1250 ml

0.5 M EDTA-8.0

200 ml

400 ml

600 ml

800 ml

1000 ml

Glucose

90 mg

180 mg

270 mg

360 mg

450 mg

 

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

STOCK

100 ml

200 ml

300 ml

400 ml

500 ml

1 M NaOH

20 ml

40 ml

60 ml

80 ml

100 ml

20% SDS

5 ml

10 ml

15 ml

20 ml

25 ml

 

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

1. Prepare bulk digestion mix using the appropriate enzyme and correct enzyme buffer.

STOCK[FINAL]Per 30 l RXN   

 

10X Buffer

1X

3.0 l

0.1 M Spermidine

2.5 M

0.75

Enzyme

25 U

up to 10.0

Plasmid (1 g/ l)

22g

22.0

  1. Add bulk mix to 500 l microfuge tube containing 20 l plasmid and incubate at 37.C for 2-3 hours or overnight. A 37.C oven works best because there is minimal condensation on the sides of the tube.

3. Stop reaction by adding 5 l 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 g/ml ethidium bromide (50 l 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/ l). 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.

PCR Protocol For Plasmids

(Suzanne Livingston, University of Minnesota)

Stock

Amount

Reaction Concentration

Plasmid DNA (20 ng)

10.0 m l
0.2 ng/m
l

Forward primer (10 ng/m l)

2.0 m l

0.2 ng/m l

Reverse primer (10 ng/m l)

2.0 m l

0.2 ng/m l

Taq Polymerase (2 units)

0.5 m l

0.02 U/m l

Taq Polymerase buffer (10X)

10.0 m l

1X

dNTPs (10 mM)

2.0 m l

200 m M

Sterile distilled H20

73.5 m l

 

Note: If MgCl2 is not in your 10X buffer, add 6 l of 25mM MgCl2 per reaction for a reaction concentration of 1.5mM and add 6 l less H20.

  1. Aliquot 10 l of DNA into a 0.5 ml microfuge tube for each PCR reaction.
  1. Determine the total number of PCR reactions.

     

  2. Multiply this number by each of the amounts in the left column of the table above. Add 1 or 2 extra reactions for pipetting error.

     

  3. Mix the resulting amounts of each stock together into a tube on ice to make a bulk reaction mixture.
  4. Pipet 90 l of the bulk reaction mixture into each 0.5 ml microfuge tube with the DNA sample. Mix well with pipet.
  5. Add 50 l of mineral oil to the top of each 0.5 ml microfuge tube (if needed).
  6. Place in PCR machine and run the following program:

    Plasmid Amplification Program

    STEP 1: 94oC 6 minutes

    STEP 2: 30 cycles of:

    95oC 1 minutes

    45oC* 2 minutes

    72oC 3 minutes

    STEP 3: 72oC 10 minutes

    STEP 4: 4oC HOLDING TEMPERATURE

    *This temperature may change up to 52oC according to the stringency required.

     

  7. After run, remove tubes from the machine and place on ice. Pipet PCR reaction out from under oil (if added in #6 above). Add 10 l of 5X SGB dye to each sample and run on an LMP gel to verify size and purify insert.

Beckmann DU-65 Spectrophotometer
DNA Quantifation Program

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 l sample in 745 l 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 l.

e. FINAL g/l?

At what concentration would you like your sample, from which this aliquot has been taken, to be diluted too? The default is 0.2 g/l.

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

Stock Solutions

100X Denhardt's

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.

 

50% Dextran Sulfate

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 l aliquots at -20.C.

 

0.5 M EDTA pH 8.0

Dissolve 186.12 g Na2EDTA2H20 (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.

 

10 mg/ml Ethidium Bromide

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 6H2O in dH2O to a final volume of 100 ml. Autoclave.

 

1 M NH4OAc

Dissolve 7.71 g ammonium acetate in dH2O to a final volume of 100 ml. Filter sterilize.

 

7.5 M NH4OAc

Dissolve 57.83 g ammonium acetate in dH2O to a final volume of 100 ml. Filter sterilize.

 

5 M NaCl

Dissolve 292.2 g NaCl in dH2O to a final volume of 1000 ml. Autoclave.

 

2.5 M NaOAc

Dissolve 20.5 g sodium acetate (anhydrous) in dH2O to a final volume of 100 ml. Autoclave.

 

1 M NaOH

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

Dissolve 268 g of sodium phosphate, dibasic, heptahydrate in dH2O to a final volume of 1000 ml. Autoclave.
 

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 -

STOCK

500 ml

1000 ml

2000 ml

5000 ml

NaH2PO4 H2O

46 g

92 g

184 g

460 g

Na2HPO4 .7H2O

45 g

90 g

180 g

450 g

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.

 

10 mg/ml RNAse A

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.

 

500 U/ml RNAse T1

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.

20% SDS

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)

STOCK

1 liter

2 liter

3 liter

4 liter

5 liter

NaCl

219g

438

657

876

1095

C6H5O7Na3 2H2O

110g

220

330

440

550

Adjust pH to 7.4 with 1 N HCl.

 

0.1 M Spermidine

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)

Stock

50 ml

100 ml

500 ml

1000 ml

2000 ml

1 M Tris pH 8.0

0.5 ml

1.0 ml

5.0 ml

10.0 ml

20.0 ml

0.5 M EDTA pH 8.0

0.1 ml

0.2 ml

1.0 ml

2.0 ml

4.0 ml

Autoclave.

 

LB Media

Per liter:

 
10 g Bacto-tryptone
5 g Bacto-Yeast extract
10 g NaCl

 

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:

 

12 g bacto-tryptone

24 g bacto-yeast extract

4 ml glycerol

 

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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