MicroGEM Quick-Start Guide

DNA Extraction UsingPDQeX
forensicGEM Universal

MicroGEM
Going to extremes foreffortless DNA extraction

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QSG_007_190531_PDQeX forensicGEM Universal

About this Guide

MicroGEM

This Quickstart Guide provides methods to extract DNA from a range of forensic sample types.
Because crime scene samples vary a great deal, we are available to provide help with developing
any specific method. Please contact us at info@microgembio.com.
Procedure overview: MicroGEM extraction products use a unique mixture of thermophilic andmesophilic enzymes. If a 52°C step is included in the method, this is to allow the mesophilic
enzyme cocktail Histosolv to weaken and degrade cellular material. The 75°C step then activatesa thermophilic proteinase that lyses the cells, kills nucleases and strips the DNA of nucleosomes.
A final 95°C step shrinks the PDQeX inner tubing, bursts the valve and pushes the extract througha column which removes the proteinase and inhibitors.

Preparation and Storage of Liquid Reagents

MicroGEM•Histosolv is a mixture of reagents that aids with digestion. It is delivered as a lyophilised
powder. This should be resuspended in DNA-free water as follows:
Kit size
Code(Rxn)		
50
100
500
1000

XFU0050
XFU0100
XFU0500
XFU1000

Volume ofwater to add
0.55 ml
1.1 ml
5.5 ml11.0 ml

Reagent storage: forensicGEM reagents are stable at room temperature but on arrival should bestored at 4°C. After tubes have been opened, the forensicGEM enzyme and the Histosolv shouldbe placed at -20°C to safeguard against accidental contamination. All buffers can remain at 4°Cfor convenience.

General Instructions

MicroGEM••••All manipulations should be performed in a clean-room or a PCR hood.
Labcoats, gloves and hairnets should be worn at all times.
Use only certified DNA-free tubes and reagents.
Wash equipment that will come into contact with the sample in 0.5% bleach. Rinse withDNA-free water.

Precautions

MicroGEM••••Do not load the PDQeX machine if the control screen indicates a temperature above 35° C.
Ensure the collection drawer and heating block are clean and DNA-free.
Ensure the collection drawer is inserted as far as possible, and that it is straight.
If fewer than 24 reactions are planned, make sure that the PCR tubes are placed in drawer
wells corresponding to the channels to be used in the heating block.

MicroGEM Reagent QC

MicroGEM

MicroGEM reagents are manufactured and quality tested following ISO 18385:2016. Our reagentsare made from certified DNA-free chemicals and solutions, and all buffers and enzymes are treatedwith DNAse and UV before shipment.

Important Technical Tips

MicroGEM•PDQeX forensicGEM Universal is a method that lyses cells and removes nucleoproteinsfrom the DNA.

•There is no concentration step in the procedure and so the concentration of the extract isdependent on: 1) The quality of the sample; 2) In the case of swabs, the type of swab andthe volume of water used to wash the swab; 3) The extraction volume (which in some cases
can be scaled).

•DNA extracted using the PDQeX forensicGEM Universal kit is suitable for High-Throughput
Sequencing (HTS) and many types of genotyping including SNP and STR analysis as well as
quantitative, multiplex and end-point PCR.

•DNA extracted using PDQeX forensicGEM Universal can be quantified using a qPCR or byusing fluorescent dyes like Pico Green, iQuant, Qubit assays or the like. Nanodrop is incompatible with PDQeX reagents.

•As with any preparative method for nucleic acid extraction, best results are obtained whensamples are handled at 4°C, or on ice, before and after extraction.

•The haem colouration carries through to the DNA leaving the sample slightly pink. This does
not cause inhibition of PCR, qPCR or human profiling.

•For long term storage of the extracted DNA, add TE buffer to 1x (10 mM Tris, pH 7.5, 1 mMEDTA) and store at -20°C.

Buccal - Liquid, Swabs and Stains

MicroGEM

The processing of the sample will vary dependent on sample type. For liquid samples, volumes
should be between 5-20 µl. With cotton swabs, add 1/4 of the swab directly to the PDQeX cartridge.
Stained fabric can be swabbed or small portions added directly to the tube.

Preparing the extraction mixture
For each extraction, make up:
10 μl

10x BLUE Buffer

2 μl  	 forensicGEM
DNA-free water - Add to a final volume of 100 µl(Note - this will vary depending on whether you add any
liquid with your sample).

PDQeX extraction
1.

Add the extraction mix with sample into each PDQeX cartridge (100 µl).

2.

Put the cap on the PDQeX cartridge by completely inserting the tapered column into
the cartridge.

3.

Load into the collection drawer either:						• 24 well plate• 8 strip tubes• Individual tubes

4.

Put the drawer in place.

5.

Insert the PDQeX cartridges into the heating block.

6.

Cover the cartridges with the hinged flap and close the sliding door.
MAKE SURE THE PDQEX CARTRIDGES CORRESPOND WITH A COLLECTION
TUBE OR WELL- OTHERWISE YOU WILL LOSE YOUR SAMPLE

7.

Select the ‘Buccal’ program for the extraction:			

75°C

5 mins95°C

2 mins•Times may be adjusted by internal laboratory optimisation.

•Changes to the default temperatures are not recommended.

Solid Tissue

MicroGEMCut the tissue into cubes of approximately 1- 2 mm3. With hair follicles, use 1-3 hairs. Cut off
the shaft 4 mm above the follicle.

Preparing the extraction mixture
For each extraction, make up:
10 μl

10x ORANGE+ Buffer

2 μl  	 forensicGEM
10 μl

Histosolv

78 μl

DNA-free water

PDQeX extraction
1.

Add the extraction mix with sample into each PDQeX cartridge (100 µl).

2.

Put the cap on the PDQeX cartridge by completely inserting the tapered column intothe cartridge.

3.

Load into the collection drawer either:						• 24 well plate• 8 strip tubes• Individual tubes

4.

Put the drawer in place.

5.

Insert the PDQeX cartridges into the heating block.

6.

Cover the cartridges with the hinged flap and close the sliding door.
MAKE SURE THE PDQEX CARTRIDGES CORRESPOND WITH A COLLECTION
TUBE OR WELL- OTHERWISE YOU WILL LOSE YOUR SAMPLE

7.

Select the ‘Tissue’ program for the extraction:			

52°C

5 mins75°C

10 mins

95°C

2 mins•Times may be adjusted by internal laboratory optimisation.

•Changes to the default temperatures are not recommended.

Saliva on Storage Cards

MicroGEM

Depending on the storage card, it is typical that the preservatives in the card are inhibitory to Taq
DNA polymerase and so a pre-wash is recommended prior to DNA extraction. For storage cards
without preservatives (e.g. MicroGEM Storage cards, Whatman 903), the pre-wash step is likely
not necessary, and may potentially cause a loss of sample. Test a pre-wash step before running
precious samples.

Pre-Wash (Optional)
1.

Remove one 3 mm disc from the card-stored sample and place into a thin-walled PCR tube.

2.

Wash the disk in 100 μl of DNA-free water by incubating at room temperature for 15 min.

3.

Aspirate the water from the disc and discard the water.

Preparing the extraction mixture
For each extraction, make up:
10 μl

10x BLUE Buffer

2 μl  	 forensicGEM
88 μl
DNA-free water		PDQeX extraction
1.

Add the extraction mix with sample into each PDQeX cartridge (100 µl).

2.

Put the cap on the PDQeX cartridge by completely inserting the tapered column into
the cartridge.

3.

Load into the collection drawer either:						• 24 well plate• 8 strip tubes• Individual tubes

4.

Put the drawer in place

5.

Insert the PDQeX cartridges into the heating block.

6.

Cover the cartridges with the hinged flap and close the sliding door.
MAKE SURE THE PDQEX CARTRIDGES CORRESPOND WITH A COLLECTION
TUBE OR WELL- OTHERWISE YOU WILL LOSE YOUR SAMPLE

7.

Select the ‘Buccal’ program for the extraction:			

75°C

5 mins95°C

2 mins•Times may be adjusted by internal laboratory optimisation.

•Changes to the default temperatures are not recommended.

Blood - Liquid, Swabs and Stains

MicroGEM

The processing of the sample will vary dependent on sample type. For liquid samples (e.g. fresh,EDTA, heparin, citrate), volumes should be between 1-10 µl. With cotton swabs, add 1/4 of theswab directly to the PDQeX cartridge. Stained fabric can be swabbed or small portions added
directly to the tube.

Preparing the extraction mixture
For each extraction, make up:
10 μl

10x ORANGE+ Buffer

2 μl  	 forensicGEM
10 μl

EnhancerDNA-free water - Add to a final volume of 100 µl(Note - this will vary depending on whether you add any
liquid with your sample).

PDQeX extraction
1.

Add the extraction mix with sample into each PDQeX cartridge (100 µl).

2.

Put the cap on the PDQeX cartridge by completely inserting the tapered column intothe cartridge.

3.

Load into the collection drawer either:						• 24 well plate• 8 strip tubes• Individual tubes

4.

Put the drawer in place.

5.

Insert the PDQeX cartridges into the heating block.

6.

Cover the cartridges with the hinged flap and close the sliding door.
MAKE SURE THE PDQEX CARTRIDGES CORRESPOND WITH A COLLECTION
TUBE OR WELL- OTHERWISE YOU WILL LOSE YOUR SAMPLE

7.

Select the ‘Blood’ program for the extraction:			

75°C

10 mins

95°C

2 mins105°C

2 mins•Times may be adjusted by internal laboratory optimisation.

•Changes to the default temperatures are not recommended.

Blood on Storage Cards

MicroGEM

Depending on the storage card, it is typical that the preservatives in the card are inhibitory to Taq
DNA polymerase and so a pre-wash is recommended prior to DNA extraction. For storage cards
without preservatives (e.g. MicroGEM Storage cards, Whatman 903), the pre-wash step is likely
not necessary, and may potentially cause a loss of sample. Test a pre-wash step before running
precious samples.

Pre-Wash (Optional)
1.

Remove one 3 mm disc from the card-stored sample and place into a thin-walled PCR tube.

2.

Wash the disk in 100 μl of DNA-free water by incubating at room temperature for 15 min.

3.

Aspirate the water from the disc and discard the water.

Preparing the extraction mixture
For each extraction, make up:
10 μl

10x ORANGE+ Buffer

2 μl  	 forensicGEM
10 μl

Enhancer

78 μlDNA-free water		PDQeX extraction
1.

Add the extraction mix with sample into each PDQeX cartridge (100 µl).

2.

Put the cap on the PDQeX cartridge by completely inserting the tapered column intothe cartridge.

3.

Load into the collection drawer either:						• 24 well plate• 8 strip tubes• Individual tubes

4.

Put the drawer in place

5.

Insert the PDQeX cartridges into the heating block.

6.

Cover the cartridges with the hinged flap and close the sliding door.
MAKE SURE THE PDQEX CARTRIDGES CORRESPOND WITH A COLLECTION
TUBE OR WELL- OTHERWISE YOU WILL LOSE YOUR SAMPLE

7.

Select the ‘Blood’ program for the extraction:			

75°C

10 mins

95°C

2 mins105°C

2 mins•Times may be adjusted by internal laboratory optimisation.

•Changes to the default temperatures are not recommended.