29th June 2024

MPC Protein Precipitation Solution

MPC Protein Precipitation Solutions are designed for use with the MasterPure™ kit protocols only.

Precipitation Protocols using MPC Protein Precipation Solution:

  1. Total Nucleic Acid Precipitation Protocol
  2. DNA Precipitation Protocol
  3. RNA Precipitation Protocol
  4. Total Nucleic Acid Precipitation from Plasma
  5. Total Nucleic Acid Precipitation from Whole Blood Lysis
  6. Total Nucleic Acids, DNA or RNA Precipitation from Buffy Coat of Blood
  7. Complete Removal of RNA

Total Nucleic Acid Purification Protocol

    1. Follow MasterPure™ Protocol for lysis of fluid, cell, tissue, whole blood sample or FFPE tissue.
    2. Add 150 µl of MPC Protein Precipitation Reagent to 300 µl of lysed sample and vortex vigorously for 10 seconds.
    3. Pellet the debris by centrifugation at 4°C for 10 minutes at =10,000 x g in a microcentrifuge.   If the resultant pellet is clear, small, or loose, add an additional 25 µl of MPC Protein Precipitation Reagent, mix, and pellet the debris again.
    4. Transfer the supernatant to a clean microcentrifuge tube and discard the pellet.
    5. Add 500 µl of isopropanol to the recovered supernatant. Invert the tube 30-40  times.
    6. Pellet the total nucleic acids by  centrifugation   at 4°C for 10 minutes in a microcentrifuge.
    7. Carefully pour off the isopropanol without dislodging the pellet.
    8. Rinse twice with 70% ethanol, being careful to not dislodge the pellet.   Centrifuge briefly if the pellet is dislodged. Remove all of the residual ethanol with a pipet.
    9. Resuspend the total nucleic acids in 35 µl of TE Buffer.

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DNA Precipitation Protocol

    1. Follow MasterPure™ Protocol for lysis of fluid, cell, tissue, whole blood sample or FFPE tissue.
    2. Add 150 µl of MPC Protein Precipitation Reagent to 300 µl of lysed sample and vortex vigorously for 10 seconds.
    3. Pellet the debris by centrifugation at 4°C for 10 minutes at =10,000 x g in a microcentrifuge. If the resultant pellet is clear, small, or loose, add an additional 25 µl of MPC Protein Precipitation Reagent, mix, and pellet the debris again.
    4. Transfer the supernatant to a clean microcentrifuge tube and discard the pellet.
    5. Add 500 µl of isopropanol to the recovered supernatant. Invert the tube 30-40 times.
    6.  Pellet the DNA by centrifugation at 4°C for 10 minutes in a microcentrifuge.

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RNA Precipitation Protocol

(A) Precipitation of Total Nucleic Acids (for all biological samples)

  1. Follow MasterPure™ Protocol for lysis of fluid, cell, tissue, whole blood sample or FFPE tissue.
  2. Add150 µl of MPC Protein Precipitation Reagent to 300 µl of lysed sample and vortex   vigorously for 10 seconds.
  3. Pellet the debris by centrifugation at 4°C for 10 minutes at =10,000 x g in a microcentrifuge. If the resultant pellet is clear, small, or loose, add an additional 25 µl of MPC Protein Precipitation Reagent, mix, and pellet the debris again.
  4. Transfer the supernatant to a clean microcentrifuge tube and discard the pellet.
  5. Add 500 µl of isopropanol to the recovered supernatant. Invert the tube 30-40 times.
  6. Pellet the total nucleic acids by centrifugation at 4°C for 10 minutes in a microcentrifuge.
  7. Carefully pour off the isopropanol without dislodging the total nucleic acid pellet.

If removal of contaminating DNA from the RNA is required, proceed with Part B. Otherwise, complete the  remainder of Part A.

8.  Rinse twice with 70% ethanol, being careful to not dislodge the total nucleic acid pellet. Centrifuge briefly if the pellet is dislodged. Remove all of the residual ethanol with a pipe.
9.   Resuspend the  total  nucleic acids in 35 µl of TE Buffer.

(B) Removal of Contaminating DNA from Total Nucleic Acid Preparations (for all biological samples)

    1. Remove all of the residual isopropanol with a pipet.
    2. Prepare 200 µl of DNase I solution for each sample by diluting 5 µl of RNase-Free DNase I up to 200 µl with 1X DNase Buffer.
    3. Completely resuspend the total nucleic acid pellet in 200 µl of DNase I solution.
    4. Incubate at 37°C for 10 minutes. Note: Additional incubation (up to 30 minutes) may be necessary to remove all contaminating DNA.
    5. Add 200 µl of 2X  T and C  Lysis  Solution;  vortex for 5 seconds.
    6. Add 200 µl of MPC Protein Precipitation Reagent; vortex 10 seconds; place on ice for 3-5 minutes.
    7. Pellet the debris by centrifugation at 4°C for 10 minutes at =10,000 x g in a microcentrifuge.
    8. Transfer the supernatant containing the RNA into a clean microcentrifuge tube and discard the pellet.
    9. Add 500 µl of  isopropanol to the supernatant. Invert the tube 30-40 times.
    10. Pellet the purified RNA by centrifugation at 4°C for 10 minutes in a microcentrifuge.
    11. Carefully pour off the isopropanol without dislodging the RNA pellet.
    12. Rinse twice with 70% ethanol,  being careful to not dislodge the pellet. Centrifuge briefly if the pellet is dislodged. Remove all of the residual ethanol with a pipet.
    13. Resuspend the RNA in 10-35 µl of TE Buffer.
    14. Add 1 µl of RiboGuard™ RNase Inhibitor (optional).

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Protocol for Precipitation of Total Nucleic Acids from Plasma:

(C) Precipitation of Total Nucleic Acids (from plasma)

  1. Follow MasterPure™ Protocol for lysis of plasma
  2. Place the samples on ice for 5 minutes
  3. Add 250 µl of MPC Protein Precipitation Reagent and vortex vigorously for 10 seconds.
  4. Pellet the debris by centrifugation at 4°C for 10 minutes at =10,000 x g in a microcentrifuge.
  5. Transfer the supernatant to a clean microcentrifuge tube and discard the pellet.
  6. Add 600 µl of isopropanol to the recovered supernatant. Invert the tube 30-40 times.
  7. Pellet the nucleic acid by centrifugation at 4°C  for 10 minutes in a  microcentrifuge.
  8. Carefully pour off  the isopropanol without  dislodging the pellet.

If  removal of DNA from RNA preparations is required, proceed with the protocol in Part D.  Otherwise complete the remainder of Part C.

9.   Rinse twice with 70% ethanol,  being careful to not dislodge the pellet.    Centrifuge briefly if the pellet is dislodged. Remove all of the residual ethanol with a pipet.
10. Resuspend the nucleic  acid in 35 µl of TE Buffer.

(D) Removal of Contaminating DNA from RNA Preparations (from plasma)

    1. Remove all of the residual isopropanol with a pipet.
    2. Prepare 200 µl of DNase I solution for each sample by diluting 10µl of RNase-Free DNase I up to 200 µl with 1X DNase Buffer.
    3. Completely resuspend the total nucleic acid pellet in 200 µl of DNase I solution.
    4. Incubate at 37°C for 10 minutes.
    5. Add 200 µl of 2X T & C Lysis Solution;  vortex for 5 seconds.
    6. Add 200 µl of MPC Protein Precipitation Reagent; vortex 10 seconds; place on ice for 3-5 minutes.
    7. Pellet the debris by centrifugation for 10 minutes at > or =10,000 x g in a microcentrifuge.
    8. Transfer the supernatant containing the RNA into a clean microcentrifuge tube and discard the pellet.
    9. Add 500 µl of  isopropanol to the supernatant. Invert the tube 30-40 times.
    10. Pellet the purified RNA by centrifugation at 4°C for 10 minutes in a microcentrifuge.
    11. Carefully pour off the isopropanol without dislodging the RNA pellet.
    12. Rinse twice with 70% ethanol,  being careful to not dislodge the pellet. Centrifuge briefly if the pellet is dislodged. Remove all of the residual ethanol with a pipet.
    13. Resuspend the RNA in 10-35 µl of TE Buffer.
    14. Add 1µl of RiboGuard™ RNase Inhibitor (optional).

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Protocol for Precipitation of Total Nucleic Acids from Whole Blood Lysis:

(F) Precipitation of Total Nucleic Acids (from whole blood)

  1. Follow MasterPure™ Protocol for lysis of Whole Blood (without RBC lysis)
  2. Place the sample on ice for 5 minutes
  3. Add 225µl of MPC Protein Precipitation Reagent and vortex vigorously for 10 seconds.
  4. Pellet the debris by centrifugation at 4°C for 10 minutes at > or = 10,000 x g in a microcentrifuge.
  5. Transfer the supernatant to a clean microcentrifuge tube and discard the pellet.
  6. Add 600µl of isopropanol to the recovered supernatant. Invert the tube 30-40 times.
  7. Pellet the nucleic acid by centrifugation at 4°C for 10 minutes in a microcentrifuge.
  8. Carefully pour off the isopropanol without dislodging the pellet.

If  removal of DNA from RNA preparations is required, proceed with the protocol in Part G.  Otherwise complete the remainder of Part F.

9.   Rinse twice with 70% ethanol,  being careful to not dislodge the pellet.    Centrifuge briefly if the pellet is dislodged. Remove all of the residual ethanol with a pipet.
10. Resuspend the nucleic acid in 35 µl of TE Buffer.

(G) Removal of Contaminating DNA from RNA Preparations (from whole blood)

    1. Remove all of the residual isopropanol with a pipet.
    2. Prepare 200 µl of DNase I solution for each sample by diluting 10µl of RNase-Free DNase I up to 200 µl with 1X DNase Buffer.
    3. Completely resuspend the total nucleic acid pellet in 200 µl of DNase I solution.
    4. Incubate at 37°C for 10 minutes.
    5. Add 200 µl of 2X T & C Lysis Solution;  vortex for 5 seconds.
    6. Add 200 µl of MPC Protein Precipitation Reagent; vortex 10 seconds; place on ice for 3-5 minutes.
    7. Pellet the debris by centrifugation for 10 minutes at > or =10,000 x g in a microcentrifuge.
    8. Transfer the supernatant containing the RNA into a clean microcentrifuge tube and discard the pellet.
    9. Add 500 µl of  isopropanol to the supernatant. Invert the tube 30-40 times.
    10. Pellet the purified RNA by centrifugation at 4°C for 10 minutes in a microcentrifuge.
    11. Carefully pour off the isopropanol without dislodging the RNA pellet.
    12. Rinse twice with 70% ethanol,  being careful to not dislodge the pellet. Centrifuge briefly if the pellet is dislodged. Remove all of the residual ethanol with a pipet.
    13. Resuspend the RNA in 10-35 µl of TE Buffer.
    14. Add 1µl of RiboGuard™ RNase Inhibitor (optional).

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Protocol for Purification of Total Nucleic Acids, DNA or RNA from Buffy Coat of Blood

(H) Precipitation of Total Nucleic Acids (from buffy coat)

  1. Follow MasterPure™ Protocol for lysis from Buffy Coat
  2. Add 300 µl of MPC Protein Precipitation Reagent and vortex vigorously for 10 seconds.
  3. Pellet the debris by centrifugation at 4°C for 10 minutes at > or =10,000 x g in a microcentrifuge.
  4. Transfer the supernatant to a clean microcentrifuge tube and discard the pellet.
  5. Add 750 µl of isopropanol to the recovered supernatant. Invert the tube 30-40  times.
  6. Pellet the nucleic acid by centrifugation at 4°C for 10 minutes in a microcentrifuge.
  7. Carefully pour off the isopropanol without dislodging the pellet.

If removal of DNA from RNA preparations is required, proceed with the protocol in Part H. Otherwise, complete the remainder of Part I.

8. Rinse twice with 70% ethanol, being careful to not dislodge the pellet. Centrifuge briefly if the pellet is dislodged. Remove all of the residual ethanol with a pipet.
9. Resuspend the total nucleic acids in 35 µl of TE Buffer.

(I) Removal of Contaminating DNA from RNA Preparations (from buffy coat)

    1. Remove all of the residual isopropanol with a pipet.
    2. Prepare 200 µl of DNase I solution for each sample by diluting 10µl of RNase-Free DNase I up to 200 µl with 1X DNase Buffer.
    3. Completely resuspend the total nucleic acid pellet in 200 µl of DNase I solution.
    4. Incubate at 37°C for 10 minutes.
    5. Add 200 µl of 2X T & C Lysis Solution;  vortex for 5 seconds.
    6. Add 200 µl of MPC Protein Precipitation Reagent; vortex 10 seconds; place on ice for 3-5 minutes.
    7. Pellet the debris by centrifugation for 10 minutes at > or =10,000 x g in a microcentrifuge.
    8. Transfer the supernatant containing the RNA into a clean microcentrifuge tube and discard the pellet.
    9. Add 500 µl of  isopropanol to the supernatant. Invert the tube 30-40 times.
    10. Pellet the purified RNA by centrifugation at 4°C for 10 minutes in a microcentrifuge.
    11. Carefully pour off the isopropanol without dislodging the RNA pellet.
    12. Rinse twice with 70% ethanol,  being careful to not dislodge the pellet. Centrifuge briefly if the pellet is dislodged. Remove all of the residual ethanol with a pipet.
    13. Resuspend the RNA in 10-35 µl of TE Buffer.
    14. Add 1µl of RiboGuard™ RNase Inhibitor (optional).

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Protocol for Complete Removal of RNA:

  1. Add 1 µl of RNase A to the sample; mix thoroughly.
  2. Incubate at 37°C for 30 minutes.
  3. Add 14 µl TE Buffer and 50 µl of 2X T and C Lysis Solution to each sample.
  4. Place the samples on ice for 3-5 minutes. Add 100 µl of MPC Protein Precipitation Reagent and mix by vortexing vigorously for 10 seconds.
  5. Pellet the debris by centrifugation at 4°C for 10 minutes at >=10,000 x g in a microcentrifuge.
  6. Transfer the supernatant to a clean microcentrifuge tube and discard the pellet.
  7. Add 200 µl of isopropanol to the recovered supernatant. Invert the tube several (30-40) times.
  8. Pellet the DNA by centrifugation at 4°C for 10 minutes in a microcentrifuge.
  9. Carefully pour off the isopropanol without dislodging the DNA pellet.
  10. Rinse twice with 70% ethanol, being careful to not dislodge the pellet. Centrifuge briefly if the pellet is dislodged. Remove all of the residual ethanol with a pipet.
  11. Resuspend the DNA in 35 µl of TE Buffer.

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Ordering:

  • Cat # MMP03750: MPC Protein Precipitation Solution 50ml
  • Cat # MMP095H: MPC Protein Precipitation Solution 500ml

Resources: