MicroGEM Quick-Start Guide

DNA Extraction Using

PDQeX
forensicGEM Sperm

MicroGEM
Going to extremes for
effortless DNA extraction

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or email
info@microgembio.com

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QSG_008_190531_PDQeX forensicGEM SpermAbout this Guide

MicroGEM

This Quickstart Guide provides methods to extract DNA from liquid sperm samples or stains.
We are available to provide help with developing any specific method. Please contact us atinfo@microgembio.com.
Procedure overview: MicroGEM extraction products use a unique mixture of thermophilic andmesophilic enzymes. At 52°C, mesophilic acrosome degrading enzymes come into play. The
75°C step then activates a 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 andpushes the extract through a column which removes the proteinase and inhibitors.

Preparation and Storage of Liquid ReagentsMicroGEM•Acrosolv is a mixture of reagents that weaken tissue cell walls. It is delivered as a lyophilised
powder. This should be resuspended in DNA-free water as follows:
Kit sizeCode(Rxn)		
50
100
500
1000

XSC0050
XSC0100
XSC0500
XSC1000

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
be stored at 4°C. After tubes have been opened, the forensicGEM enzyme and the Acrosolv
should be placed at -20°C to safeguard against accidental contamination. The ORANGE+ buffer
can remain at 4°C for 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 Sperm is a method that lyses cells and removes nucleoproteins fromthe 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 Sperm kit is suitable for High-Throughput
Sequencing (HTS) and many types of genotyping including SNP and STR analysis as well asquantitative, multiplex and end-point PCR.

•DNA extracted using PDQeX forensicGEM Sperm can be quantified using a qPCR or by using
fluorescent dyes like Pico Green, iQuant, Qubit assays or the like. With mixed samples, aY-specific qPCR is recommended. 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.

•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.

Extraction method

MicroGEM

Stains and Swabs
The processing of the sample will vary dependent on sample type. For liquid samples, try to keep
the volume of the liquid below 10 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 ORANGE+ Buffer

2 μl  	 forensicGEM
10 μl

Acrosolv
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 ‘Sperm’ program for the extraction:			

52°C

5 mins75°C

3 mins95°C

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

•Changes to the default temperatures are not recommended.