Instruction Manual

Table of Contents

Introduction
General Protocol for 1.5 ml Tube Size
General Protocol for 15 ml Tube Size
General Protocol for 3 ml Syringe

Application Protocols

Recovery of DNA from LMP Agarose
M13/Phagemid DNA Extraction
Lambda DNA Extraction
Total RNA Isolation
Genomic DNA Isolation
Mouse Tail Genomic DNA Isolation
RNA-Free Plasmid DNA Isolation

References

Introduction

PHASE-Guard™ is an inert proprietary gel intended for use in optimizing the recovery of nucleic acids from various standard organic extraction mixtures.¹ The use of this product in your protocol can result in the recovery of 20-30% more nucleic acid. PHASE-Guard™ is stable for at least one year when stored as described.

After organic extraction, it is often difficult to recover nucleic acid in the aqueous upper phase free from the denatured protein at the aqueous and organic phase interface. PHASE-Guard™ present during phenol, phenol-chloroform, and chloroform-isoamyl alcohol extractions migrates under centrifugal force to form a seal between the organic and aqueous phases. The organic phase and the interface material are effectively trapped below PHASE-Guard™. The barrier is sufficiently durable that the aqueous upper phase, containing the nucleic acid, can then be recovered quantitatively by simply decanting or pipetting to a fresh tube.

PHASE-Guard™ is inert, stable to heating, and does not interfere with standard nucleic acid restriction and modification enzymes. In fact, many of the reactions can be carried out in the presence of PHASE-Guard™ at the appropriate temperature and then terminated by the addition of Phenol or Phenol-Chloroform and extracted. PHASE-Guard™ can be present during the heat inactivation of enzymes (65°C for 10 minutes) prior to the organic extraction.

PHASE-Guard™ Light can be used to improve the recovery of DNA fragments from Low Melting Point (LMP) Agarose with only minor changes to the standard protocol¹ . The use of PHASE-Guard™ Heavy in a microprep plasmid DNA purification protocol allowed for the direct screening of inserts as well as probe synthesis and/or DNA sequencing within one day.² PHASE-Guard™ light may be used in the standard protocols for the preparation of plasmid DNA from E coli, phagemid DNA from M13-type phage² and phage DNA from lamda³ . PHASE-Guard™ Heavy may be used to prepare partially-purified plasmid DNA from E coli and for the preparation of total RNA by homogenization in Guanidium Isothiocyanate⁵ and organic extraction^5,10 . PHASE-Guard™ Light also may be used for isolating high molecular weight genomic DNA from blood, cultured cells, tissue¹¹, and mouse tail.

The ability of PHASE-Guard™ to separate the phases is dependent upon the composition of the aqueous and the organic media. Salt and protein in the aqueous phase have an effect on both the aqueous and organic phase densities, while different organic phase formulations also vary in density. For optimum phase separation, the compositions of the aqueous phase, organic phase, and PHASE-Guard™ must be compatible. As a result, PHASE-Guard™ is offered in two different density formulations: Light (L) and heavy (H). Please consult the table below for the formulation which fits your application.

PHASE- Guard ™ SYRINGE GENERAL PROTOCOL

1. Without putting pressure on the plunger, twist off the orange cap and discard. Screw on the supplied grey dispensing tip to the syringe and tighten securely.

2. Apply firm pressure on the plunger to dispense the PHASE-Guard™.

3. Store the assembled syringe by pulling back on the plunger slightly. It is recommended that the grey tip be left in place, as PHASE-Guard™ is quite slippery and can prevent adequate securing of the tip in future.

   NOTE: The following amounts of PHASE-Guard™ are recommended for optimum phase separation:

   approximately 100 µL PHASE-Guard™ for 0.65 mL microcentrifuge tubes;
   approximately 300 µL PHASE-Guard™ for 1.5 mL microcentrifuge tubes;
   approximately 2 mL PHASE-Guard™ for disposable 15 mL screw-cap centrifuge tubes;
   approximately 5 mL PHASE-Guard™ for disposable 50 mL screw-cap centrifuge tubes.

4. Follow the appropriate general protocol above.

RECOVERY OF DNA FROM LOW MELTING POINT AGAROSE¹ (with three PHASE-Guard™ Light 1.5 ml tube)

Materials Required:

• 3 PHASE-Guard™ Light 1.5 ml tubes
• Low Melting Point Agarose
• Tris-Acetate-EDTA (TAE) Buffer (10 mM Tris-Cl, 1 mM EDTA, pH 8.0)
• Ethidum Bromide
• TE pH 8.0 (10 mM Tris-Cl, pH 8.0, 1 mM EDTA)
• Phenol-buffer-saturated, pH 7.9
• Phenol-Chloroform-Isoamyl Alcohol 25:24:1
• Chloroform-Isoamyl Alcohol 24:1
• 10 M Ammonium Acetate
• 100% Ethanol
• Microcentrifuge tubes

1. Resolve the DNA fragments on a Low Melting Point (LMP) Agarose (Molecular Biology Grade) gel in 1X Tris-Acetate-EDTA (TAE) buffer. Do not use Tris-Borate-EDTA (TBE) as borate-gels are much more difficult to solubilize.

2. Stain the gel(s) with Ethidium Bromide, visualize with a longwave UV light, and carefully cut out the band(s) of interest with a sharp razor blade. (NOTE: Wear gloves when handling Ethidium Bromide and stained gels.)

3. Weigh a 1.5 ml PHASE-Guard™ Light tube and centrifuge at 12,000 x g for 20-30 seconds.

4. Transfer gel slice to the pre-spun, pre-weighted PHASE-Guard™ tube and determine the weight of the slice.

5. Add a volume (in µL) of TE, pH 8.0 equivalent to 5X the weight (in mg) of the slice and melt the slice in the TE, pH 8.0 at 65°C for 5-10 minutes.

6. Mix well by inversion to ensure LMP agarose slice is fully dissolved. Allow the dissolved sample to come to room temperature and then add an equal volume of room temperature buffer-saturated Phenol, pH 7.9 to the sample. Mix until homogeneous. PHASE-Guard™ will not become part of the suspension.

7. Centrifuge at full speed (12,000 xg or greater in a microcentrifuge) for 2 minutes to separate the phases. NOTE: If the resulting aqueous phase still appears cloudy, repeat the extraction with an equal volume temperature buffer-saturated Phenol, pH 7.9.

8. Recover the aqueous phase to a fresh PHASE-Guard™ Light 1.5 ml tube and extract with an equivalent volume of room temperature Phenol-Chloroform-Isoamyl Alcohol (PCI, 25:24:1). DO NOT VORTEX.

9. Centrifuge as in step 7 above. Recover the aqueous phase to a fresh PHASE-Guard™ Light 1.5 ml tube, and extract with an equivalent volume of room temperature Chloroform-Isoamyl Alcohol (CI, 24:1).

10. Centrifuge as in step 7 above and recover the aqueous phase to a suitably sized microcentrifuge tube.

11. Add 0.25 volume of 10 M Ammonium Acetate and 2.5 volume of 100% Ethanol to the sample and mix well.

12. Incubate at room temperature for 20 minutes, pellet the DNA by centrifugation, wash the pellet several times with cold 70% Ethanol, air-dry the pellet, and resuspend in a suitable buffer.

M13/PHAGEMID DNA EXTRACTION PROTOCOL (with one PHASE-Guard™ Light 15 ml tube)

Materials Required:

• 1 PHASE-Guard™ Light 15 ml tube
• 15 ml Conical centrifuge tubes
• Microfuge tubes
• 20% PEG/2.5 M NaCl
• TE pH 8.0 (10 mM Tris-Cl, pH 8.0, 1 mM EDTA)
• Phenol-water-saturated
• Phenol-Chloroform-Isoamyl Alcohol (25:24:1)
• Chloroform-Isoamyl Alcohol (24:1)
• Ethanol-3 M Sodium Acetate pH 5.2 (25:1)
• 70% Ethanol
• 100% Ethanol

The efficiency of the standard protocols^8,9 for production of single-stranded DNA may be improved through the use of PHASE-Guard™.

1. Propagate M13 phase by one of the standard procedures:
   1. For producing M13 ssDNA, we routinely mix 2.5 mL plating bacteria (5-7 hours, 50 mL culture; stored at 4°C for no more than 3 days) with 300 µL M13 phase stock (1 x 10¹¹ pfu/mL) and incubate 5 minutes at room temperature to allow phase to interact with the bacterial phage receptors. The phage-bacteria culture is added to 250 mL LB broth and incubated for 5-7 hours at 37°C with sufficient agitation for good aeration of the culture.

   2. For producing phagemid ssDNA, we routinely suspend a colony of phagemid-transformed bacteria in a 15 mL culture tube containing 3 mL LB Broth. Helper phage M13K07 is added to a final concentration of 2 x 10⁷ pfu/mL and the culture is incubated for 1.5 hours at 37°C with sufficient agitation (200-300 rpm) for good aeration of the culture. Kanamycin (25 mg/mL in water) is then added to a final concentration of 70 µg/mL. Incubation of the culture is continued overnight (12-15 hours) at 37°C.

2. Recover the entire culture volume to a centrifuge tube or bottle and pellet the bacteria by centrifugation at 4,000 xg (r_max) for 20 minutes, 4°C. Recover the resultant supernatant to a fresh tube or bottle and re-centrifuge as above.

3. Recover the resultant supernatant to a fresh centrifuge tube, add 0.25 volume of 20% PEG/2.5 M NaCl to the supernatant, mix thoroughly by repeated inversion. Incubate 20 minutes at room temperature. Centrifuge at 16,000 x g (r_max), 4°C, for 20 minutes and discard the resultant supernatant. Allow any traces of supernatant to drain from the tube.

4. Resuspend the pellet in 50 µL TE, pH 8.0 for every 1 mL of supernatant (step 3) and transfer the entire sample to a pre-spun (1500 xg for 1-2 minutes) PHASE-Guard™ Light 15 ml tube.

5. Add an equal volume of water-saturated Phenol to the sample in the PHASE-Guard™ tube, and mix thoroughly by repeated inversion. Centrifuge at 1500 xg for 2 minutes to separate the phases. [The PHASE-Guard™ will not become part of the mixture.]

6. Add an equal volume of Phenol-Chloroform-Isoamyl Alcohol (PCI, 25:24:1) to the sample in the same PHASE-Guard™ tube and extract as in step 5 above.

7. Add an equal volume of Chloroform-Isoamyl Alcohol (CI, 24:1) to the sample in the same PHASE-Guard™ tube and extract as in step 5 above.

8. Quantitatively transfer the final aqueous phase to a 2 mL microcentrifuge tube, add 3 volumes of a 25:1 Ethanol- 3M Sodium Acetate solution, and mix well.

9. Freeze at -80°C for 15-30 minutes or until frozen, thaw, and centrifuge for 20 minutes at 12,000 xg or greater.

10. Wash pellet once or twice with 0.5 mL ice cold 70% Ethanol, once with 0.5 mL ice cold 95-100% Ethanol and air-dry.

11. Resuspend the pellet in 20-50 µL TE, pH 8.0.

LAMBDA DNA EXTRACTION PROTOCOL (with one PHASE-Guard™ Light 15 ml tube)

Materials Required:

• 1 PHASE-Guard™ Light 15 ml tube
• 13 x 100 mm culture tubes
• L-broth with Mg⁺²
• SM Buffer (0.1 M NaCl, 8 mM MgSO₄, 50 mM Tris-Cl, pH 7.5, 0.01% Gelatin)
• Agarose
• l Dilutent (10 mM Tris-Cl, pH 7.5, 98 mM MgSO₄)
• DNase I
• RNase
• 20% SDS
• Proteinase K
• Phenol-Chloroform-Isoamyl Alcohol (25:24:1)
• Chloroform-Isoamyl Alcohol (24:1)
• 3 M Sodium Acetate, pH 5.2
• 70% Ethanol
• 100% Ethanol
• TE, pH 8.0 (10 mM Tris-Cl pH 8.0, 1 mM EDTA)

We have found this method to produce good quality l DNA with yields of 15-20 µg l gt10 DNA per 450 µL of supernatant processed. PHASE-Guard™ tubes also can be used in conjunction with other l DNA purification methods.

1. Grow a 10 mL stock of suitable plating bacteria in L-broth plus Mg⁺² overnight at 37°C.

2. Dilute the l phage stock to 10⁵ pfu per 0.1 mL in SM buffer. Mix 0.1 mL of the l dilution with 0.1 mL plating bacteria in a sterile 13 x 100 mm culture tube and allow phage to adsorb to the bacteria for 20 minutes at 37°C.

3. Add 2.5 mL pre-warmed (45-50°C) L-broth plus Mg⁺² containing 0.6% agarose to the tube prepared in step 2. Mix well by inversion and pour contents onto a prepared solid 100 mm L plus Mg⁺² plate. Allow the top agarose to harden at room temperature and then incubate at 37°C for 8 hours. Plaques should be evident at this time.

4. Chill the plate for 15 minutes at 4°C. Add 3 mL l dilutent to the plate and incubate overnight at 4°C with gentle rocking.

5. Collect the phage-containing l dilutent to a centrifuge tube and centrifuge at 4000 xg for 10 minutes, 4°C, to pellet the debris.

6. Transfer 450 µL of the resultant supernatant to a pre-spun (1500 xg for 1-2 minutes) PHASE-Guard™ Light 15 ml tube, add 4.5 µL 1 mg/mL DNase I and 2.0 µL 40 U/µl RNase. Mix by inversion and incubate for 30 minutes at 37°C.

7. Add 11.5 µL 20% SDS and 4.5 µL 10 mg/mL Proteinase K to the sample, mix, and incubate 30 minutes at 37°C.

8. Extract the sample twice with 500 µL Phenol-Chloroform-Isoamyl Alcohol (PCI, 25:24:1) and once with 500 µL Chloroform-Isoamyl Alcohol (CI, 24:1), centrifuge for 2 minutes at 1500 xg to separate the phases. Perform all steps in the same PHASE-Guard™ tube. [The PHASE-Guard™ will not become part of the mixture.]

9. Collect the final aqueous phase and transfer to a fresh 1.5 mL microcentrifuge tube, add 45 µL 3 M Sodium Acetate, pH 5.2 and 500 µL 100% Isopropanol, mix and incubate at room temperature for 15 minutes.

10. Centrifuge 20 minutes at 12,000 xg or greater, discard the supernatant and wash the DNA pellet two times with 70% Ethanol and once with 95-100% Ethanol prior to drying at 30°C.

11. Resuspend the DNA in 20-50 µL TE, pH 8.0.

TOTAL RNA ISOLATION PROTOCOL^5,6 (with two PHASE-Guard™ Heavy 12 ml tubes)

Materials Required:

• 2 PHASE-Guard™ Heavy 12 ml Tubes
• Guanidine Thiocyanate Solution (GIT) (4 M guanidine thiocyanate, 25 mM Sodium Citrate pH 7.0, 1 M b-mercaptoethanol)
• Tissue/Cell Homogenizer
• 2 M Sodium Acetate, pH 4.0
• Phenol-water-saturated
• Chloroform:Isoamyl Alcohol (49:1)
• Phenol-Chloroform-Isoamyl Alcohol (25:24:1)
• 100% Isopropanol
• 70% Ethanol
• 100% Ethanol
• RNase-Free Water

1. Sample Preparation:
   1. To extract RNA from Tissue, add 1.0 mL 4 M GIT to 100 mg minced tissue.

   2. To extract RNA from washed and pelleted cultured cells, add 1.0 mL 4 M GIT to 1 x 10⁷ cells.

   3. To directly extract RNA from cultured cells growing in monolayer, add 1.0 mL 4 M GIT directly to the culture dish.

2. Homogenize the tissue ( 1 A) and pelleted cells (1 B) with a Polytron, a Dounce homogenizer, or other suitable homogenization apparatus. Homogenize monolayer cells (1 C) by pipetting the mixture up and down several times, taking care to "wash" cell material free from the culture dish in the process. Use of a cell scraper will aid in collecting the homogenized monolayer cells.

3. Transfer all of the homogenate to a prespun (1500 xg for 1-2 minutes) PHASE-Guard™ Heavy 12 ml tube.

4. Add 0.1 mL 2.0 M Sodium Acetate, pH 4.0 to the sample, cap the PHASE-Guard™ tube and mix briefly by inversion.

5. Add 1.0 mL water-saturated Phenol to the sample, cap the PHASE-Guard™ tube, and mix thoroughly by repeated inversion. The PHASE-GUARD™ will not become part of the mixture. DO NOT VORTEX.

6. Add 0.3 mL Chloroform:Isoamyl Alcohol (CI, 49:1) to the sample in the same PHASE-Guard™ tube and mix thoroughly by repeated gentle inversion. DO NOT VORTEX.

7. Incubate on ice for 10 minutes.

8. Centrifuge at 4500 xg (r_max) for 5 minutes to separate the phases.

9. Transfer the aqueous (upper) phase to a fresh PHASE-Guard™ Heavy 12 ml tube which as been pre-spun as in step 3 above.

10. Add 1.0 mL Phenol-Chloroform-Isoamyl Alcohol (PCI, 25:24:1) to the aqueous phase in the new PHASE-Guard™ tube. Mix thoroughly by repeated gentle inversion. DO NOT VORTEX.

11. Centrifuge at 4500 xg (r_max) for 5 minutes to separate the phases.

12. Collect the resultant aqueous phase to an RNase-free centrifuge tube. Add an equal volume of 100% isopropanol, securely insert the RNase-free stopper, and mix by repeated inversion.

13. Incubate at -20°C for 20-30 minutes and then centrifuge at 16,000 xg for 30 minutes at speed.

14. Discard the resultant supernatant and wash the RNA pellet twice with 5 mL 70% Ethanol, using 2-3 minutes at 16,000 xg to re-pellet the sample. NOTE: Samples may be stored in the 70% Ethanol wash at this stage at -70°C or colder for extended periods.

15. Wash the pellet once with 5 mL 95%-100% Ethanol as in step 14 above.

16. Discard the final wash and dry the pellet at room temperature. Gentle heating at 30°C will speed up the drying process.

17. Re-dissolve the pellet in a suitable volume (200 µL) of RNase-free ultrapure water. Store the RNA solution at -70°C. Absorbance determinations should be performed in RNase-free TE, pH 8.

GENOMIC DNA ISOLATION PROTOCOL (with two PHASE-Guard™ Light 15 ml tubes)

Materials Required:

• 2 PHASE-Guard™ Light 15 ml tubes
• 15 ml conical centrifuge tubes
• Tris Buffered Saline (TBS:50 mM Tris-Cl pH 7.5, 200 mM NaCl, 3 mM KCl, 0.02% Sodium Azide)
• 2X Lysis Buffer (0.65 M Sucrose, 20 mM Tris-Cl, pH 8.0, 10 mM MgCl2, 2% Triton X-100)
• Saline/EDTA (75 mM NaCl, 24 mM EDTA)
• Proteinase K in 10 mM Tris-Cl, pH 8, 1.0 mM CaCl2, 30% Glycerol
• 10% SDS
• Water Saturated Phenol
• 2 M KCl
• 95% Ethanol
• 70% Ethanol
• TE (10mM Tris-Cl pH 8.0, 1 mM EDTA)

Genomic DNA is fragile. High molecular weight DNA is sheared easily by mechanical forces. DO NOT VORTEX SOLUTIONS CONTAINING GENOMIC DNA.

1. Isolation of Nuclei.

   NOTE: An equivalent amount of homogenized tissue may be substituted as starting material.

   1. Blood - CAUTION: Wear suitable protective apparel and take appropriate safety measures when working with human blood.
      1. Transfer 5 mL whole blood (containing EDTA as the anti-coagulant) to a 15 mL polypropylene centrifuge tube. NOTE: When working with avian blood, or blood from other species that have nucleated red blood cells, use 0.2 mL blood and 4.8 mL 1X Tris-Buffered Saline instead of 5 mL blood.

      2. Add 5 mL 2X Lysis Buffer and mix by gentle inversion.

      3. Incubate solution for 5 minutes on ice.

      4. Pellet the nuclei by centrifugation at 1000 x g for 12 minutes at 4°C.

      5. Decant the supernatant and drain any residual supernatant by inverting the tube on a paper towel for 2 minutes. NOTE: The pelleted nuclei may be stored at -70°C for several weeks at this point, however, higher molecular weight DNA is obtained when genomic DNA is isolated from freshly prepared nuclei.

      6. Proceed to Section II below.

   2. Tissue Culture Cells
      1. Suspend cells (equivalent of 100 mM plate) in the growth medium and transfer to a 15 mL polypropylene centrifuge tube.

      2. Pellet the cells by centrifugation at 250 x g for 5 minutes at 4°C.

      3. Decant the supernatant and drain any residual supernatant by inverting the tube on a paper towel for 2 minutes.

      4. Suspend the cells in 1 mL 1X TBS by repeated up and down pipetting, then add 4 mL 1X TBS and 5 mL 2X Lysis Buffer to the cell suspension and mix by inversion.

      5. After a 5 minute incubation on ice, pellet the nuclei by centrifugation at 1100 x g for 12 minutes at 4°C.

      6. Decant the supernatant and drain any residual supernatant by inverting the tube on a paper towel for 2 minutes. NOTE: The nuclei may be stored at -70°C for several weeks at this point, however, high molecular weight DNA is obtained from freshly prepared nuclei.

      7. Proceed to Section II below.

2. Lysis of Nuclei and Deproteinization of DNA
   1. Suspend the pelleted nuclei in 2 mL Saline/EDTA Solution by repeated up and down pipetting.

   2. Carefully transfer suspended nuclei to a pre-spun (1500 xg for 2-3 minutes) PHASE-Guard™ Light 15 mL tube.

   3. Add 50 µL 20 mg/mL Proteinase and 200 µL 10% SDS to the suspended nuclei and mix by gentle inversion.

   4. Incubate for 2 hours at 37°C with occasional gentle mixing. NOTE: This incubation may be extended to 16-18 hours if that is more convenient.

   5. Proceed to Section III below.

3. Extraction of Protein
   1. Add 4 mL water-saturated Phenol to the PHASE-Guard™ tube containing the deproteinized DNA. Cap the tube tightly.

   2. Mix by shaking vigorously enough to form a homogeneous suspension. DO NOT VORTEX. The PHASE-Guard™ will not become part of the mixture.

   3. Centrifuge at 1500 x g for 5 minutes to separate the phases.

   4. Carefully decant upper aqueous phase containing the DNA into a fresh, pre-spun, PHASE-Guard™ Light 15 ml tube.

   5. Repeat steps 1 through 3 but this time in step 1 extract with 4 mL water-saturated Phenol-Chloroform (PC, 1:1).

   6. Carefully decant the upper phase containing the DNA into a clean 15 mL polypropylene centrifuge tube.

   7. Proceed to Section IV below.

4. Precipitation of DNA

   At this point, the DNA should have been extracted with both Phenol and Phenol:Chloroform and should be in a clean 15 mL polypropylene screw cap centrifuge tube.

   1. Add 100 µL 2 M KCl and mix by gentle inversion.

   2. Overlay the DNA solution with 5 mL 95% Ethanol by slowly pipetting the Ethanol down the side of the tube.

   3. Place a Pasteur pipet tip at the interface of the DNA-Ethanol solution and spool the DNA onto the pipet tip by swirling the pipet, keeping the tip at the interface, until the 2 phases are completely mixed.

   4. Place the pipet tip with the spooled DNA in 1 mL 70% Ethanol for about 2 minutes.

   5. Remove the pipet from the 70% Ethanol and hold upright (tip up) for a few seconds to allow the excess Ethanol to drain away. Do not allow the DNA to dry.

   6. Set the pipet tip in a microcentrifuge tube containing 200 µL TE, pH 8 and incubate for 20 minutes at room temperature. The DNA should slide from the pipet into the TE. If necessary, gently remove the DNA from the pipet by scraping the DNA onto the interior of the tube.

   7. Redissolve the DNA completely by incubating overnight at 4°C. Store the DNA at 4°C. DO NOT FREEZE.

5. Determination of Genomic DNA Concentration.
   1. Add 10 µL of the redissolved DNA to 490 µL 0.1 N NaOH. Vortex the diluted DNA solution.

   2. Read the absorbance at 260 nm against a 0.1 N NaOH blank.

   3. To calculate DNA concentration use the formula:
      Absorbance at 260 nm X 2.5 = _____ mg DNA per mL.

MOUSE TAIL GENOMIC DNA ISOLATION PROTOCOL (with two PHASE-Guard™ Heavy 1.5 mL tubes)

Materials Required:

• 2 PHASE-Guard™ Heavy 1.5 ml tubes
• Tail Buffer (50 mM Tris-Cl, pH 8.0, 100 mM EDTA, 100 mM NaCl, 1% SDS)
• Proteinase K
• RNase A
• Phenol:Chloroform:Isoamyl Alcohol (PCI 25:24:1)
• Chloroform-Isoamyl Alcohol (24:1)
• 100% Isopropanol
• 70%, 95% EtOH
• TE (10 mM Tris-Cl, pH 8.0, 1 mM EDTA)

1. Place a 1 cm tail sample into a 1.5 mL microcentrifuge tube; this may be stored at -20°C. To minimize possible cross-contamination, do not mince the sample. Add 700 µL Tail Buffer to the sample.

2. Add 35 µL 10 mg/mL Proteinase K to the sample and mix briefly.

3. Incubate at 55-60°C overnight with mixing (a hybridization oven works well). This step should result in the complete solubilization of the tail fragment. In the case of incomplete digestion, more Proteinase K can be added and the samples incubated for several more hours.

4. Add 20 µL 10 mg/mL RNase A (DNase-free) to the sample. Mix briefly and incubate at 37°C for 1-2 hours.

5. Transfer the entire solution to a pre-spun (1,500 xg for 1-2 minutes) 1.5 mL PHASE-Guard™ Heavy tube.

6. Add 0.5 mL Phenol-Chloroform-Isoamyl Alcohol (PCI, 25:24:1) to the sample in the PHASE-Guard™ Heavy 1.5 mL tube and mix well by repeated inversion. DO NOT VORTEX.

7. Centrifuge at full speed (12,000 x g or greater) for 5 minutes in a microcentrifuge, then carefully transfer the resultant aqueous phase to a fresh pre-spun PHASE-Guard™ Heavy 1.5 mL tube.

8. Add 0.5 mL Chloroform-Isoamyl Alcohol (CI, 24:1) to the sample in the PHASE-Guard™ tube and mix well by repeated inversion. DO NOT VORTEX.

9. Centrifuge at full speed (12,000 x g or greater) for 5 minutes, then carefully transfer the resultant aqueous phase to a fresh microcentrifuge tube.

10. Fill the sample-containing tube with 100% Isopropanol and mix thoroughly by repeated inversion. DO NOT VORTEX. A visible DNA precipitate should form. Proceed immediately to step 11.

11. Recover the DNA precipitate by touching it to a heat-sealed glass micropipette tip or by lifting the DNA with a yellow pipette tip and partial suction from a pipettor. Transfer the DNA to a 1.5 mL microcentrifuge tube containing 70% Ethanol. If the DNA is not stringy, pellet by a brief, low speed centrifugation.

12. Wash DNA with the 70% Ethanol, then wash twice with 95% Ethanol.

13. Allow the DNA to partially dry and then either transfer the DNA to a microcentrifuge containing 400 µL TE, pH 8.0 or add 400 µL TE, pH 8.0 to the DNA in the tube. DO NOT VORTEX OR RE-PIPET TO RESUSPEND DNA.

14. Resolubilize the DNA by placing the sample tube in a rack on a horizontal rotator set at 30-60 rpm and rotate overnight. Resolubilization may be facilitated by heating the samples at 50°C.

15. Remove the glass pipet tip if present and continue resolubilization on a end-over-end rotator for 2 hours.

16. Determine the DNA concentration by measuring the absorbance of a 1:20 dilution. When pipetting DNA, shearing can be minimized either by using large-bore pipet tips or by cutting off part of the pipet tip to increase the diameter of the tip opening.

RNA-FREE PLASMID DNA ISOLATION PROTOCOL (with one PHASE-Guard™ Light 15 ml tube)

Materials Required:

• 1 PHASE-Guard™ Light 15 ml tube
• Solution A   25 mM Tris-Cl pH8.0, 10 mM EDTA
• Solution B   0.2 N NaOH, 1.0% SDS
• Solution C   7.5 M Ammomium Acetate
• TE 10 mM Tris-Cl pH8.0, 1 mM EDTA
• 100% Isopropanol
• RNase (40 U/µl)
• Phenol:Chloroform:Isoamyl Alcohol (25:24:1)
• PCI 25:24:1 + 0.1% 8 hydroxyquinoline
• Ethanol

Rapid Preparation of Cleared Lysate:

This rapid version of modified^1,2 alkaline lysis procedure^4,5 works well with the pZ523 plasmid purification spin column. The following protocol is for use with amplified and 12-14 hour non-amplified 1 liter cultures grown for no longer than 12-14 hours in 0.5 liter of enriched growth media such as superbroth or terrific broth (TB). The reagents used in the cleared lysate preparation should be scaled down proportionately for smaller culture volumes down to 200 mL.

1. Pellet the bacteria from the culture at 10,000 xg (r_max) for 5 minutes at 2-4°C.

2. Resuspend the bacterial pellet in a total of 30 mL Solution A. Pipet up and down or vortex as necessary to fully resuspend the bacteria.

3. Add 30 mL room temperature Solution B to the suspension. Mix thoroughly by repeated gentle inversion. DO NOT VORTEX. Lysate should become essentially uniform and translucent.

4. Add 30 mL ice-cold Solution C to the lysate. Mix thoroughly by repeated gentle inversion. DO NOT VORTEX. A flocculent precipitate should appear.

5. Centrifuge at 16,000 x g (r_max) for 30 minutes at 2-4°C.

6. Carefully remove the supernatant to a fresh centrifuge bottle. Do not carry over any whitish-grey pellet material. Floating pellet material may be removed onto the surface of a disposable glass or plastic pipet.

7. Add 54 mL (0.6 X volume) room temperature 100% Isopropanol to the supernatant. Mix thoroughly by repeated inversion. DO NOT VORTEX.

8. Centrifuge at 16,000 x g (r_max) for 30 minutes at 20°C. Discard the resultant supernatant.

9. Add 25 mL 70% Ethanol to the pellet. Mix by repeated inversion and centrifuge at 16,000 x g (r_max) for 1-2 minutes to re-pellet the DNA.

10. Discard the resultant supernatant and wash the pellet with 25 mL 95-100% Ethanol as described in step 9 above.

11. Discard the resultant supernatant, carefully aspirate any excess Ethanol, and briefly dry the pellet at 30-37°C for 10-15 minutes.

12. Proceed to RNA Removal Procedure.

RNA Removal Procedure:

1. Add 1.5 mL TE, pH 8 containing 15 µL RNase (40 U/µL) to the pellet and mix gently to dissolve the pellet.

2. Centrifuge for 1-2 minutes at 10,000 x g (r_max) to consolidate sample, then transfer sample to a pre-spun (1500 xg for 1-2 minutes) PHASE-Guard™ Light 15 ml tube and proceed to step 3. NOTE: 5-10 µL of this suspension can be analyzed by 1% agarose gel electrophoresis to confirm that plasmid DNA is present.

3. Incubate for 15 minutes at 37°C in a water bath.

4. Extract sample once with 2.0 mL Phenol-Chloroform-Isoamyl Alcohol (PCI, 25:24:1 containing 0.1% 8-Hydroxyquinoline). Thoroughly mix the aqueous and organic phases by repeated inversion. DO NOT VORTEX. Centrifuge at 1500 x g (r_max) for 2 minutes at speed to separate the phases. The PHASE-Guard™ will not become part of the mixture and will form a barrier between the aqueous and organic phases during the centrifugation.

5. Add 2.0 mL PCI to the aqueous sample in the same PHASE-Guard™ tube and extract as in step 4 above. DO NOT VORTEX.

6. Add 2.0 mL Chloroform-Isoamyl Alcohol (CI, 24:1) to the aqueous sample in the same PHASE-Guard™ tube and extract as in step 4 above. DO NOT VORTEX.

7. Carefully transfer the resultant aqueous phase (1.4-1.5 mL) to a suitable fresh tube(s) and process or precipitate the sample(s) as per your protocols and procedures.

REFERENCES

1. 1. Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. "Molecular Cloning, A Laboratory Manual", 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. pp. 6.30-6.31. 2.

2. Emanuel, J.R. 1992. Nucleic Acids Res. 20(3):625. 3.

3. Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. "Molecular Cloning, A Laboratory Manual", 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. pp. 4.44-4.48. 4.

4. Chirgwin, J.M. et al. 1979. Biochem. 18(24):5294-5299. 5.

5. Chomczynski, P. and Sacchi, N. 1987. Anal. Biochem. 162:156-159. 6.

6. Birnboim, H.C. and Doly, J. 1979. Nucl. Acids Res. 7:1513-1522. 7.

7. Birnboim, H.C. 1988. Nucl. Acids Res. 16(4):1487-1497. 8.

8. Current Protocols in Molecular biology, Volume I. 1989. Ausubel, F.M. et al.. (eds.), John Wiley and Sons, New York, NY pp. 1.14.1 - 1.15.4. 9.

9. Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. "Molecular Cloning, A Laboratory Manual", 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. pp. 4.29 - 4.32. 10.

10. Sabourin, C.L.K., et al.. 1996. Int. J. Oncol. 8:49-56. 11.

11. Laws, G.M. and S.P. Adams. 1996. BioTechniques 20:36-38. 12.

12. Murphy, N.R. and R. J. Hellwig. 1996 BioTechniques 21(5):934-939.

For Research Only