M AINE S TATE P OLICE C RIME L ABORATORY: F ORENSIC B IOLOGY S ECTION
MICROCON-100™/CENTRICON-100™ CONCENTRATION OF DNA
PRINCIPLE :
Both the Microcon-100 and Centricon-100 centrifugal filter devices concentrate solutions of DNA by ultrafiltration in a fixed-angle rotor centrifuge. They are used to concentrate, desalt, and/or remove low molecular weight macromolecules from DNA extracts. When very low yields of DNA are suspected after the extraction procedure, the total volume of
DNA solution can be reduced in order to increase the amount of DNA capable of being added to the DNA amplification and typing reactions. They are also used as a final clean-up step following phenol/chloroform/isoamyl alcohol organic extractions.
SPECIMEN:
Any aqueous solution believed to contain a low concentration of DNA.
Aqueous phase of DNA extraction from organic extractions.
REAGENTS AND SPECIAL SUPPLIES:
Bleach, 10% (7 mM sodium hypochlorite solution)
Centricon-100 concentration tubes
Microcon-100 concentration tubes
Tips, aerosol-resistant (e.g., for P-10, P-100, P-1000 pipettes)
Tris-EDTA (TE
-4
), (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA)
Tubes (1.7 ml)
INSTRUMENTATION AND EQUIPMENT:
Clinical centrifuge (e.g., Jouan C412)
Microcentrifuge (e.g., Eppendorf 5415C)
Pipettes (e.g., P-10, P-100, P-1000)
QUALITY ASSURANCE:
1.
Extractions of specimens are to be performed in the extraction laboratory only.
2.
Ensure that the following worksheet is completed for this procedure and placed in the appropriate case folder: "Microcon-100™/Centricon-100™ Concentration of
DNA.”
3.
Ensure that all reagents satisfy the minimum standards for quality control.
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MICROCON-100™/CENTRICON-100™ CONCENTRATION OF DNA
4.
Ensure that all equipment satisfies the minimum standards for quality control, where appropriate.
5.
In order to prevent contamination, all steps in this procedure should be conducted in the proper laboratory hoods when appropriate.
6.
Any laboratory workspace and all pipettes and racks to be used in this procedure must be cleaned with 10% bleach and thoroughly dried before beginning. When using a laminar flow hood, turn on the ultraviolet light for a minimum of 10 minutes after cleaning.
7.
Before use, forceps, scissors, etc., must be cleaned with 10% bleach, rinsed in ethanol, and dried thoroughly. In addition, they should be cleaned in the same manner following the processing of each individual specimen (when used).
8.
Microcon and Centricon components are not autoclaveable; do not autoclave or irradiate.
9.
To prevent contamination, the TE
-4 buffer (in 50 ml conical tubes) must be irradiated with 6 j/cm
2
. Follow "Irradiation of Reagents and Supplies in the
Ultraviolet Crosslinker" SOP for appropriate method of irradiation.
10.
No reagents or supplies used for the extraction of DNA from specimens will be allowed in a post-amplification room.
11.
Any equipment taken from a post-amplification room to a pre-amplification room must be sterilized with 10% bleach immediately before removal from the post- amplification room and immediately upon arrival in the pre-amplification room.
Reagents should never be transferred from a post-amplification room to a preamplification room.
12.
Only one evidence specimen will be open at any one time.
13.
Change pipette tips between each transfer or addition of sample or reagent, unless otherwise noted.
14.
No aliquot of any reagent may be returned to the original stock container.
15.
A minimum of one reagent blank must be carried throughout the extraction procedure and assayed in parallel with the evidence samples.
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M AINE S TATE P OLICE C RIME L ABORATORY: F ORENSIC B IOLOGY S ECTION
MICROCON-100™/CENTRICON-100™ CONCENTRATION OF DNA
SAFETY:
1.
WARNING - CHEMICAL HAZARD. Wear chemical-resistant gloves and eye protection when handling phenol-chloroform solution. Use in well ventilated area.
2.
All appropriate MSDS sheets must be read prior to performing this procedure.
3.
Treat all biological specimens as potentially infectious. Gloves, safety glasses, and a laboratory coat must be worn at all times. Follow Universal Precautions.
4.
Avoid direct exposure to ultraviolet light when using the germicidal lamp in the biological hood.
5.
Distinguish all waste as general, biohazard, or Sharps
TM
and discard appropriately.
PROCEDURAL NOTES:
1.
Wipe down work area with 10% bleach. Use gloves and aerosol pipette tips to minimize contamination. Change or clean gloves and instruments between each sample.
2.
DNA extracted with Chelex is single-stranded and should be quantitated by slot blot since it is unsuitable for quantitation methods that use ethidium bromide.
PROCUDURE:
Microcon-100 concentrator:
1.
Assemble and label a Microcon-100 concentration device for each sample to be extracted (including Reagent Blanks) by inserting a “sample reservoir” into open
“retentate/filtrate vial” (for filtration). Close the caps on the assemblies and label the
“sample reservoir” with case number and item number. Also label an extra
“retentate/filtrate vial” for each sample (for recovery).
2.
Carefully transfer the entire DNA extract to the “sample reservoir” (upper chamber) of the Microcon-100 assembly (up to 0.5 ml can be transferred at once). Cap tightly with attached lid.
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MICROCON-100™/CENTRICON-100™ CONCENTRATION OF DNA
NOTE: Avoid touching the membrane with the pipette tip. Avoid transferring any phenol/chloroform/isoamyl alcohol or Chelex resin beads to the Microcon-100
“sample reservoir.”
3.
Centrifuge the tubes at 2400 rpm (500 x g) in an Eppendorf microcentrifuge for at least 15 minutes. Repeat centrifugation step until almost all liquid is filtered through the membrane, leaving only a residue of liquid at the edge.
NOTE: If liquid is slow to pass through the filter, the filter may be clogged (in which case the retentate should be transferred to a new Microcon-100 sample reservoir), the DNA solution may be too concentrated (in which case the sample may be quantitated at whatever volume has been achieved), or the sample may be too cold to pass through the filter efficiently (in which case the sample needs to warm up to room temperature).
4.
If filtering an organic extraction: discard the filtrate (liquid that has passed through the filter into the “retentate/filtrate vial”), add 0.5 ml of TE -4
buffer to the “sample reservoir” and repeat the centrifugation in step 3 above. Repeat wash and centrifugation steps twice for a total of three washes.
5.
Place sample reservoir upside down in the second labeled “retentate/filtrate vial” (see step 1 above). Spin for 3 minutes at 3500 rpm (1000 x g), or pulse briefly, to transfer retentate from filter into new vial.
6.
Bring retentate up to volume with TE
-4
buffer if necessary, for a final volume of as little as 20
 l, or as much as 200
 l. The sample is now ready for quantitation and the
PCR amplification process.
7.
Store at 2-8 o
C for short-term storage. Freeze at -20 o
C (or colder) for long-term storage.
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MICROCON-100™/CENTRICON-100™ CONCENTRATION OF DNA
Centricon-100 concentrator:
1.
Assemble and label a Centricon-100 concentration device for each sample to be extracted (including Reagent Blanks) by inserting a “sample reservoir” into a “filtrate vial” (for filtration). Place a “retentate vial” on the top of the “sample reservoir” to close the assembly, and label the “retentate vial” with case number and item number.
Also label an extra microcentrifuge tube for each sample (for final storage).
2.
Carefully transfer the entire DNA extract to the “sample reservoir” (upper chamber) of the Centricon-100 assembly (up to 2 ml of aqueous phase can be transferred at once).
NOTE: Avoid touching the membrane with the pipette tip. Avoid transferring any phenol/chloroform/isoamyl alcohol or Chelex resin beads to the Centricon-100
“sample reservoir.”
3.
Centrifuge the tubes at 2400 rpm (500 x g) in a clinical centrifuge for at least 15 minutes. Repeat centrifugation step until almost all liquid is filtered through the membrane, leaving only a residue of liquid at the edge
NOTE: If liquid is slow to pass through the filter, the filter may be clogged (in which case the retentate should be transferred to a new Centricon-100 sample reservoir), the DNA solution may be too concentrated (in which case the sample may be quantitated at whatever volume has been achieved), or the sample may be too cold to pass through the filter efficiently (in which case the sample needs to warm up to room temperature).
4.
If filtering an organic extraction: add 2 ml of TE
-4
buffer to the “sample reservoir” and repeat the centrifugation in step 3 above. Repeat wash and centrifugation steps twice for a total of three washes.
5.
Turn “retentate vial” and “sample reservoir” upside down in the “filtrate vial.” Spin for 3 minutes at 3500 rpm (1000 x g), or pulse briefly, to transfer retentate from filter into the “retentate vial.”
6.
Transfer retentate to extra microcentrifuge tube (see step 1 above) for final storage.
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M AINE S TATE P OLICE C RIME L ABORATORY: F ORENSIC B IOLOGY S ECTION
MICROCON-100™/CENTRICON-100™ CONCENTRATION OF DNA
7.
Bring retentate up to volume with TE
-4
buffer if necessary, for a final volume of as little as 20
 l, or as much as 200
 l. The sample is now ready for quantitation and the
PCR amplification process.
8.
Store at 2-8 o C for short-term storage. Freeze at -20 o C (or colder) for long-term storage.
REFERENCES :
Package Insert, Millipore Microcon Centrifugal Filter Devices User Guide.
Package Insert, Millipore Centricon Centrifugal Filter Devices User Guide.
Reviewed
Supervisor, Forensic DNA Section
Date
Adopted
Annual Review:
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Reviewed
Director, Crime Laboratory
Date
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