DNA Diagnostic Laboratory - Human and Clinical Genetics (LUMC) - LMDp_Protocols

Phenol extraction of DNA solutions

(last modified January 18, 2000)


Protocol

for the purification of DNA solutions using a phenol extraction (kindly provided by Michiel van der Wielen and Els Voorhoeve)

NOTE: chloroform has recently been replaced by dichloormethane


Target group

The technicians who are working in the clinical DNA-diagnostic department.


Responsibilities

The technicians who perform the DNA-diagnostic analysis of specific diseases are responsible for purification of unpure DNA solutions according to this protocol.


Definitions


Description

In this protocol the purification of unpure DNA solutions according to the phenol extraction procedure [1] is described. Starting material can be unpure DNA samples or unpure cell lysates, both isolated according to the protocol for DNA isolation from blood (ALG001).

Sometimes it is necessary to purify DNA solutions prior to further enzymatic manipulations. This is important when DNA solutions contain contaminants that will inhibit restriction enzyme digestion (ALG 004) or PCR amplification. One of the most commonly used methods for de-proteinising a DNA solution is extraction with phenol, which efficiently denatures proteins and probably dissolves denatured protein. dichloormethane stabilises the rather unstable boundary between an aqueous phase and a pure phenol layer. The use of a mixture of dichloormethane and phenol also reduces the amount of aqueous solution retained in the organic phase, compared to a pure phenol phase, to maximise the yield. Iso-amyl alcohol is added to prevent foaming of the solution and to aid in the separation of the organic and aqueous phases. The denatured protein forms a layer at the interface between the aqueous and organic phases and is thus isolated from the bulk of the DNA in the aqueous layer. Following the phenol extraction a 2-propanol precipitation is performed to concentrate the DNA solution. The procedure takes place in a pre-PCR lab. The DNA-samples are stored in the cold room. DNA-samples should never enter the post-PCR rooms.


Durables

NOTE: equivalent durables may be used


Consumables

NOTE: equivalent consumables may be used


Chemicals

NOTE: equivalent consumables may be used


Solutions

For the solutions marked with a * a special form containing the protocol is filled out. The other solutions are ready to use or made according to the protocols described here.


Rules

General administration

Safety measures


Phenol extraction protocol

Since the starting material for this procedure can vary between DNA samples obtained after completion of the procedure "DNA isolation from blood" (ALG001), and DNA solutions obtained after cell lysis (day 2 DNA isolation from blood - ALG001), the procedure is divided into two different protocols.

  1. EXTRACTION OF DNA SAMPLES
    1. estimate the volume of the DNA sample using a P1000. If the sample contains more then 750 l, divide the sample into two equal portions. A volume of less then 500 l should always be completed to 500 l with autoclaved Elga water
    2. transfer the DNA sample to an 1.5 ml eppendorf tube
    3. add 500 l of phenol:dichloormethane:iso-amyl alcohol and put the tube(s) in the top over for 10 min
    4. centrifuge for 10 min at 14,000 rpm
    5. using a P200 pipet with a Cell Saver tip, transfer the upper aqueous phase into an 1.5 ml eppendorf tube
      NOTE: avoid the transfer of debris from the interface
    6. repeat steps 3, 4 and 5 if a lot of debris is visible in the interface. Make a remark of this repetition in WFENOL-form
    7. add an equal volume of dichloormethane:iso-amyl alcohol, mix the phases by inverting the tube(s) for 30 seconds
    8. centrifuge for 5 min at 14,000 rpm and transfer the upper aqueous phase to an eppendorf tube without disturbing the interface
    9. add 1/20 volume of 3.0 M NaAc (pH5.6) and mix by inverting the tube
    10. precipitate the DNA with one volume of 2-propanol
    11. collect the precipitated DNA with a sealed Pasteur pipet
      NOTE: DNA from one individual can be collected with the same Pasteur pipet (pooling of samples)
    12. wash the DNA in 70% ethanol and dissolve the DNA in 100-500 l TE-4 in a cryotube with a printed label (see administration)
    13. to inactivate possible DNases, incubate the tube for 30 minutes in the 65C incubator
    14. put the tube(s) overnight on the rollerbench (RT)
    15. store the dissolved DNA sample in the cold room
      NOTE: if the sample is not well dissolved, add more TE-4 and place the tube back on the rollerbench until the DNA is dissolved completely
    16. complete the WFENOL-form
  2. EXTRACTION OF CELL LYSATES
    1. add 1.5 ml of autoclaved Elga water to the cell lysate
      NOTE: samples of one individual can be pooled
    2. write the DNA-numbers on both the side and the cap of the 50 ml blue cap tubes
    3. add an equal volume (5 ml) of phenol:dichloormethane:iso-amyl alcohol with a P5000 and put the tube(s) in the top over for 10 min.
    4. centrifuge for 15 min at 3,000 rpm
    5. transfer the upper aqueous phase into a blue cap tube
      NOTE: use a P1000 and cut the end of the blue tip, or use a plastic Pasteur pipet
      NOTE: avoid the transfer of debris from the interface
    6. repeat steps 3, 4 and 5 if a lot of debris is visible in the interface
      NOTE: make a remark of this repetition in the WFENOL-form
    7. add an equal volume of dichloormethaan:isoamyl alcohol and mix the phases by inverting the tube(s) several times
    8. centrifuge for 5 min at 3,000 rpm and transfer the upper aqueous phase,without disturbing the interface, to a blue cap tube
    9. add 1/20 volume of 3.0 M NaAc (pH5.6) and gently homogenise the solution
    10. precipitate the DNA with one volume of 2-propanol
    11. collect the precipitated DNA with a sealed Pasteur pipet
    12. wash the DNA in 70% ethanol and dissolve it in 100-500 l TE-4 in a cryotube with a printed label (see administration)
    13. to inactivate possible DNases, incubate the tube for 30 min in the 65C incubator
    14. put the tube(s) overnight on the rollerbench (RT)
    15. store the dissolved DNA sample in the cold room
      NOTE: add more TE-4 if the sample is not well dissolved and place the tube back on the rollerbench until the DNA is dissolved completely
    16. complete the WFENOL-form and store it in the folder WFENOL

References

[1] Ausubel F.M., Brent R., Kingston R.E., Moore D.D., Seidman J.G., Smith J.A., Struhl K. (1987). Current Protocols In Molecular Biology, Volume 1, Wiley Interscience. Chapter 1.2.1



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