MicroGEM Quick-Start Guide

DNA Extraction Using
prepGEM 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_001_190531_prepGEM Universal

prepGEM
prepGEM is for DNA extractions from a range of sample types.
More information can be found at 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.

When your kit arrives
The Histosolv is delivered as a dry powder. Add DNA-free water
as follows.
Kit size
Code(Rxn)		
50
100
500
1000

PUN0050
PUN0100
PUN0500
PUN1000

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

Reagent storage
prepGEM reagents are stable at room temperature but onarrival should be stored at 4°C. After tubes have been opened,
the prepGEM should be placed at -20°C to safeguard against
accidental contamination. The buffer can remain at 4°C forconvenience.
Procedure overview

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 of prepGEM
Make sure the suspension is agitated prior toadding
3. Incubate at:
75°C for 5 minutes
95°C for 2 minutes
Mix before using
DO NOT CENTRIFUGE.
The DNA is high molecular 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 CultureprepGEM DNA extraction is ideal for low cell numbers. Because thebuffers used for prepGEM are compatible with most downstream
processes, it means that the entire sample can be used. In addition,
because prepGEM does not need purifications steps, extractions
can be performed in sub-microlitre volumes.
With cultured cells, you can expect linear yields for 5 to approx
100,000 cells and is ideal for single-cell work. For low numbersof cells we recommend reducing the extraction volume. Theminimum volume possible will depend on evaporation with theequipment you are using.
The recommended amounts of prepGEM to use for different
extraction volumes are below. Use 1/10th volume of 10X BLUEbuffer.
Extraction Volume

Cell numbers

Volume of prepGEM

50 - 100 µl
20 - 50 µl
5 - 20 µl
1 - 15 µl

50,000 - 500,000
5000 - 50,000
100 - 5000
1 - 500

1 µl
1 µl
0.5 µl
0.2 µlSample handling will vary with different sample types. An outlineof some suggested procedures is provided below. More information
is available at www.microgembio.com.
Handling different culture types
Cells in suspension
1.
Centrifuge the suspension at 200 x g for 5 mins.
2.
Remove all of the liquid.
3.
Resuspend the pellet in prepGEM extraction reagents.

Tissue Culture
Handling different culture types (continued)
Adherent cells
If the cells are in flasks, dislodge cells by preferred method (Trypsinor cell scraper) and centrifuge suspension at 200 x g for 5 mins.
Otherwise, the prepGEM reagents can be added directly to theadhered layer.
1.
2.

Remove all of the liquid.
Add prepGEM extraction reagents.

Cells stored in RNAlater™
1.
2.		
3.

Centrifuge suspension at 3,000 x g for 5 mins.
Remove all of the liquid (a quick spin on a bench
centrifuge can help to gather the last few drops).
Resuspend the pellet in prepGEM extraction reagents.

Cell pellets
Up to 5 x 105 cells can be extracted using the recommended method.
Linear extraction efficiency is best achieved within the range of<10 cells to approximately 105. Cell pellets can be used directly.
Alternatively, the pellet can be resuspended in 1X BLUE buffer and an
appropriate quantity added to the extraction.
FACS and LCM
Cells can be collected directly in the extraction reagent mastermix
or the reagents added directly to a capillary from LCM. If cells arecollected in a different buffer, it may be necessary to add 1/10th
volume of the prepGEM buffer after collection. We recommend
using prepGEM reagents within one hour of preparation. For
longer periods, reagents should be frozen.
prepGEM is sensitive to EDTA and other chelating agents. Ifcells are presented in EDTA-containing solutions, they should becentrifuged at 200 x g and washed in 1X BLUE buffer before use.

Tissue Culture
Extraction (50 µl reaction - can be scaled to any volume)
1. Add:								Cell suspension or pellet
5 µl 10x BLUE Buffer
1 µl prepGEM
Water to a final volume of 50 µl

2. Vortex and incubate:
75 °C for• >50,000 cells -10 min• 1,000 - 50,000 cells - 5 min• <1,000 cells - 2 min
95 °C for 2 min
4 °C HOLD
A thermal cycler can be used for this step.
3. Add 1/10th volume of 10x TE Buffer and store at -20°C or below.

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 prepGEM		
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.

Insects and Mouse Tails
Extraction Method1.

Add to the material (volumes can be scaled
up/down for bigger/smaller starting samples):		
44 µl of PCR grade water,		
5 µl of 10x BLUE Buffer		
1 µl prepGEM
2. Incubate at:
75 °C for 15 minutes
95 °C for 2 minutes
A thermal cycler can be used for this step
3. Transfer supernatent to a new tube
The DNA is in this solution. Do not discard.
The sample is now ready for PCR.
For storage, at TE to 1x and store at -20°X

Centrifugation is undesirable for automation andshould not normally be needed. However withsome material, two minutes at 5,000 x g may
assist in clarifying the extract. Note: fast spins cansediment genomic DNA.
Depending on the age and quality of tissue, 0.1- 5 µl of extract is recommended for a 25 µl PCR

Saliva 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
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 water by
incubating at room temperature for 15 min.
Aspirate the water from the disc and discard thewater.
3. Add to the tube:						5 µl of 10x BLUE Buffer
44 µl of DNA-free water
1 µl prepGEM

3. Incubate in a thermal cycler:		
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 MicroGEM blood 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
3,000 r.c.f. should be spun for 10 minutes. Centrifugation shouldbe 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/prepgem-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 prepGEM		
Add 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 ofa 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 prepGEM
4. Incubate in a thermal cycler:		
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 punchThe sample is now ready for quantification.
Typically, 2 - 5 µl should be used in PCRTechnical Tips
•prepGEM 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 prepGEM 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.

prepGEM reagents are stable at room temperature, but after
the tubes have been opened and for longer term storage, theenzymes should be stored at -20°C and the buffers at 4°C.

More information can be found at :www.microgembio.com
If you still need help, email us at:
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_001_190531_prepGEM Universal