MicroGEM Quick-Start Guide

DNA Extraction Using
forensicGEM Universal

MicroGEM
Going to extremes for
effortless DNA extraction

Find more information atwww.microgembio.com
or email
info@microgembio.com

MicroGEM International PLC: Research Use Only. All products are subject
to a limited use license. See the product documentation on our website.

QSG_003_190531_forensicGEM Universal

forensicGEM
forensicGEM is validated for forensic DNA extractions. Validation
data can be obtained from www.microgembio.com.
General instructions••••

All manipulations should be performed in a clean-room ora 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 0.05% bleach. Rinse thoroughly with DNA-free water.

Histosolv 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 size
Code(Rxn)		
50
100
500
1000

FUN0050
FUN0100
FUN0500
FUN1000

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 and the Histosolv should beplaced at -20°C to safeguard against accidental contamination.
The buffer can remain at 4°C for convenience.
Procedure overview: MicroGEM extraction products use aunique mixture of thermophilic and mesophilic enzymes.
Where a low temperature stage is used, mesophilic cell wall
degrading enzymes come into play. The 75° step then activatesa thermophilic proteinase that lyses the cells, kills nucleases andstrips the DNA of nucleosomes. A final 95° step deactivates thethermophilic proteinase.

Buccal swabs
1. Wash the buccal swab in the minimum
amount of DNA-free water to cover the swab.
Typically, a cotton swab requires 400-500 µl.
Use a rolling action against the tube sides andpress the swab against the side to squeeze as
much of the liquid as possible.
An alternative approach is to cut off a portionof the swab.
2. In a thin-walled PCR tube add:		
20 µl of the eluate.		
10 µl of 10x BLUE Buffer 		
69 µl of DNA free water		
1 µl forensicGEM
Make sure the suspension is agitated prior toadding
3. In a thermal cycler, incubate:				

75°C for 5 minutes
95°C for 2 minutes

Mix before using
DO NOT CENTRIFUGE. The DNA is highmolecular weight and can be sedimented
with high speed centrifugation.
The sample is now ready for analysis.
Typically, the method yields DNA at 0.5 - 2 ng / µl dependingon the quality of the sampling and the size of the swab.

Tissue
Solid Tissue
Cut the tissue into cubes of approximately 1- 2 mm3. With hairfollicles, use 1-3 hairs. Cut off the shaft 4 mm above the follicle.

1. Mix in a thin-walled PCR tube:
79 µl DNA-free water.
10 µl of 10x ORANGE+ Buffer 		
1 µl forensicGEM
10 µl Histosolv
2. Add the sample
3. Mash the sample with a pipette tip anddisperse by vortexing.
4. In a thermal cycler, incubate:						52°C for 5 minutes
75°C for 10 minutes
95°C for 3 minutes

3. Aspirate the extract away from residualmaterial.
The DNA is in this solution. Do not discard.
For long term storage of the extracted DNA,add one tenth volume 10x TE buffer (100 mMTris, pH 7.5, 10 mM EDTA). Store at -20°C.

Cigarette Butts
Cigarette butts are difficult to process because of the release oftars and phenolics that can inhibit Taq DNA polymerase. The
forensicGEM method resolves this problem by using a gentle lysis
which extracts the DNA without releasing the inhibitors.
1. Remove a 1 cm strip of paper from thecigarette butt and cut into quarters. Thesecan be processed separately as replicates.
2. Cut each sample of paper into smaller
pieces (3 mm squares) and place in a thinwalled PCR tube or 96-well PCR tray.
3. Add:		
44 µl DNA-free water.		
5 µl of 10x BLUE Buffer		
1 µl forensicGEM
4. In a thermal cycler, incubate:		
75°C for 5 minutes		
95°C for 2 minutes
5. Aspirate the extract away from the paper
immediately and transfer to a fresh tube.
The DNA is in this solution. Do not discard.
The paper can be discarded.
Please see the cigarette application note at:
http://www.microgembio.com/products/forensicgem-universal/

Saliva on Storage Cards
Depending on the storage card, it is typical that the preservatives inthe card are inhibitory to Taq DNA polymerase and so a pre-wash isrecommended prior to DNA extraction
1. Remove one 3 mm disc from the card-stored
sample and place into a thin-walled PCR tube
or a 96-well plate.
Uneven application of the swab onto the storage
card results in DNA yield variations. For the best
results, punch in the centre of the area where
the sample was applied.
2. Wash the disk in 100 µl of DNA-Free waterby incubating at room temperature for 15 min.
Aspirate the water from the disc and discard thewater.
3. Add to the tube:						44 µl of DNA-free water
5 µl of 10x BLUE Buffer
1 µl forensicGEM

3. In a thermal cycler, incubate:		
75°C for 5 minutes		
95°C for 2 minutes
4. Pipette the solution to a new tube
The DNA is in this solution - not the punch.

The sample is now ready for quantification.
Typically, 2 - 5 µl should be used in PCR

Blood Methods
Centrifugation Tips
The forensicGEM buffer is a proprietary formulation thatprecipitates PCR inhibitors. The solid material should not be
disturbed when removing the supernatant.
Typically, 5 minutes at 13,000 r.c.f is sufficient to give a well-packedpellet. Longer spins should be used for lower r.c.f. centrifugations.
For example, a typical 96-well plate, swing out rotor rated at 3,000r.c.f. should be spun for 10 minutes. Centrifugation should be
performed immediately after extraction.
Notes•

Yields will vary depending on the WBC count of the sample.

•Information on how to optimise blood DNA extraction canbe found on our website at:
http://www.microgembio.com/products/forensicgem-universal/•You should be aware that haem coloration carries throughto the DNA leaving the sample slightly pink. This does notcause inhibition of PCR, qPCR or human profiling.

Liquid Blood
Extraction Method
1. In a thin-walled PCR tube add:		
2-5 µl of liquid blood		
10 µl of 10x RED+ Buffer		
1 µl forensicGEM		
DNA-free water to 100 µl
2. In a thermal cycler, incubate:		
75°C for 5 minutes
95°C for 5 minutes
3. Centrifuge in a microcentrifuge at fullspeed for 5 min
SEE CENTRIFUGATION TIPS
4. Pipette the supernatant to a new tube
without disturbing the pellet
This solution contains the DNA.
Do not discard.
The sample is now ready for use. Typically, 5 µl of
a 1:5 dilution gives the best results in a PCR or HID
profiling, but depending on your application, weadvise testing a few different dilutions.
Yields of ~0.5 ng/µl can be expected from fresh blood.

Please visit www.microgembio.com for more information

Blood on Storage Cards
Depending on the storage card, it is typical that the preservatives
in the card are inhibitory to Taq DNA polymerase and so a prewash is recommended prior to DNA extraction
1. Remove one 3 mm disc from the card-stored
blood sample and place into a thin-walled
PCR tube or a 96-well tray. For the best results,
punch in the centre of the area where theblood was applied.
2. Wash the disk in 100 µl of DNA-Free water by
incubating at room temperature for 15 min.
Aspirate the water from the disc(s) and discard.
3. In a thin-walled PCR tube add:		
5 µl of 10x RED+ Buffer		
44 µl of DNA-free water		
1 µl forensicGEM
4. In a thermal cycler, incubate:		
75°C for 5 minutes		
95°C for 5 minutes
3. Centrifuge for 2 minutes at maximum speed
and transfer the supernatant to a fresh tube
(SEE CENTRIFUGATION TIPS)The DNA is in the solution - not the punch

The sample is now ready for quantification.
Typically, 2 - 5 µl should be used in PCR

Bloodstains
You can use the same method for the storage card with bloodstains.
Methods will vary depending on the sample type.
Direct extraction
1. Cut a piece of stained fabric no bigger than 3 mm x 3 mm. And
place in a thin-walled PCR tube.
2. Add:		
5 µl of 10x RED+ Buffer		
44 µl of DNA-free water		
1 µl forensicGEM
3. In a thermal cycler, incubate:		
75°C for 5 minutes		
95°C for 5 minutes
4. Centrifuge for 2 minutes at maximum speed and transfer thesupernatant to a fresh tube
(SEE CENTRIFUGATION TIPS)
Wet swab lift
1. Lift the blood from the stain using a damp swab. Small foamswabs are preferred.
2. To a thin-walled PCR tube, add:		
10 µl of 10x RED+ Bufferr		
89 µl of DNA-free water		
1 µl forensicGEM
3

Wash the swab in the extraction solution using a rolling action
on the sides of the tube. Squeeze as much liquid as possible
out of the swab.

4. In a thermal cycler, incubate:		
75°C for 5 minutes		
95°C for 5 minutes
3. Centrifuge for 2 minutes at maximum speed and transfer thesupernatant to a fresh tube
(SEE CENTRIFUGATION TIPS)

Technical Tips•

forensicGEM is a preparative method for DNA extraction.
The method lyses cells and removes nucleoproteins fromthe DNA. Extracted DNA can be used for many types ofgenotyping including SNP and STR analysis as well as
quantitative, multiplex and end-point PCR.

•There is no concentration step in the procedure and so theconcentration of the extract is dependent on: 1) The qualityof 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 forensicGEM is largely single-stranded
because of the 95°C heat step.

•For accurate yield assessment, a qPCR is recommended.
If standard fluorescent chelating dyes are to be used fornormalising samples, then we recommend taking a sampleof the extract before the 95°C step. Alternatively, you cangenerate a standard curve using a previously-made extractthat has been quantified.

•As with any preparative method for nucleic acid extraction,
best results are obtained when samples 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 mM EDTA) and store at -20°C.
The forensicGEM reagents are stable at room temperature, but
after tubes have been opened and for longer term storage, theenzymes should be stored at -20°C and the buffers at 4°C.
More information is available on our website at:www.microgembio.com