MicroGEM
Going to extremes foreffortless DNA extraction

PLANT and Fungal DNA EXTRACTION(for use with phytoGEM kits)

MicroGEM International PLC: Research Use Only. All products are subject to alimited use license. See more information at www.microgembio.com
QSG_009_190531_PDQeX phytoGEM

DNA Extraction Protocol
Reagent Storage:••••

Histosolv*
prepGEMGREEN+ Buffer.
Enhancer.

-20° C-20° C
4° C
4° C(Limit freeze/thaw cycles)* The Histosolv is delivered as a dry powder. When your kit arrives add
DNA-free water as follows:
Kit size(Rxn)		

CodeVolume ofwater to add

50100
500
1000

PPP0050
PPP0100
PPP0500
PPP1000

0.55 ml
1.1 ml
5.5 ml11.0 ml

PRECAUTIONS
1.

Do not load the PDQeX machine if the control screen indicates atemperature above 40° C.

2.

Ensure the collection drawer and heating block are clean and DNAfree.

3.

Ensure the collection drawer is inserted as far as possible, and that
it is straight.

4.

If fewer than 24 reactions are planned, make sure that the PCR tubes
are placed in drawer wells corresponding to the channels to be usedin the heating block.

This QuickStartGguide provides methods for three sample types. Notehowever, that the phytoGEM system can be easily adapted for other sampletypes. Please contact us at...

info@microgembio.com... for advice from our team on how you can modify the methods for yourspecific sample.

For more information, visit: www.microgembio.com

METHOD I.

Extraction of DNA from storage cards

READ THE TECHNICAL TIPS AT THE END OF THIS DOCUMENT
Sample Types:••

Plant leaves, stems and roots
Plant pathogens.

Method:1.

Completely thaw prepGEM and Histosolv, and mix bygently inverting the tubes. Remove GREEN+ buffer and theEnhancer from the refrigerator and mix.

2.

Prepare the following extraction mixture:
10 µl
2 µl
10 µl
10 µl
68 µl

GREEN+ buffer
prepGEM
Histosolv
EnhancerDNA-free water3.

Dispense 100 µl of master mix into each PDQeX cartridge.

4.

Take a sample by crushing the plant onto the storage cards
using the crusher tool.
IF USING MULTI-USE PUNCHES, RINSE THE PUNCH IN DILUTE
BLEACH AND THEN ETHANOL.. BLOT DRY

5.

Take 1 - 4 punches from your storage card and drop it inthe PDQeX cartridge. Make sure that the punches arecompletely submerged in the reagents.

6.

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

7.

Load 24-well plate, 8-strip tubes or individual tubes into thecollection drawer and put the drawer in place.

8.

Insert the PDQeX cartridges into the heating block.

9.

Cover the cartridges with the hinged flap and close thesliding door.
MAKE SURE THE PDQeX CARTRIDGES CORRESPOND WITH A
COLLECTION TUBE OR WELL - OTHERWISE YOU WILL LOSEYOUR SAMPLE

10. Select the “Plant” program.
52 °C
75 °C
95 °C
••

5 mins.
5 mins. 		
2 mins.

Times may be adjusted by internal laboratory optimisation.
Changes to the default temperatures are not recommended.

METHOD 2.

Extraction of DNA from homogenised leaves,ground plant tissue

READ THE TECHNICAL TIPS AT THE END OF THIS DOCUMENT
Sample Types:••

Homogenised plant leaves, stems and roots
Plant pathogens in homogenised samples

Method:1.

Weigh 5-10 mg of leaf and place in a 1.6 ml tube. Add 100 µlof ultrapure water to this and homogenise using a sterile
hand-held homogenising tool, tissue lyser or a mortar andpestle.
Ensure the sample is homogenised completely. For groundbark, woody tissue or roots, make a suspension of 5 - 10 mg
ground tissue in 100 µl ultrapure water. Prepare and manage
on ice or at 4 °C for best results.

2.

Prepare the following extraction mixture:			 10 µl		 2 µl		10 µl		10 µl		63 µl		5 µl

10x GREEN+ buffer
prepGEM
Histosolv
Enhancer
Ultrapure water
Leaf homogenate or tissue suspension

Cut the ends off the pipette tips to help transfer thehomogenate. Mix thoroughly. Ensure large pieces of leaf
matter are removed, they will clog the extraction cartridges.
3.

Mix by vortexing and dispense into a PDQeX cartridge.

4.

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

5.

Load 24-well plate, 8-strip tubes or individual tubes into thecollection drawer and put the drawer in place.

6.

Insert the PDQeX cartridges into the heating block.

7.

Cover the cartridges with the hinged flap and close thesliding door.
MAKE SURE THE PDQeX CARTRIDGES CORRESPOND WITH A
COLLECTION TUBE OR WELL - OTHERWISE YOU WILL LOSEYOUR SAMPLE

8.

Select the “Plant” program.
52 °C
75 °C
95 °C
••

5 mins.
5 mins.
2 mins.

Times may be adjusted during method optimisation.
Changes to the default temperatures are not recommended.

METHOD 3. Extraction of DNA from fungal cultures
READ THE TECHNICAL TIPS AT THE END OF THIS DOCUMENT
Method:1. Transfer a small amount of fungal culture into a 1.6 ml
Eppendorf tube using an inoculation loop or tease tool (halfa loopful of sample is usually sufficient for good DNA yield).
Add 200 µl of ultrapure water to this and homogenise using asterile hand-held homogenising tool or tissue lyser. Prepareand manage on ice or at 4 °C for best results.
2. Prepare the following extraction mixture:
10 µl
2 µl
10 µl
10 µl
68 µl

10x GREEN+ buffer
prepGEM
Histosolv
Enhancer
Fungal homogenate

3. Mix by vortexing and dispense into a PDQeX cartridge.
4. Put the cap on the PDQeX cartridge by completely inserting
the tapered column into the cartridge.
5. Load 24-well plate, 8-strip tubes or individual tubes into thecollection drawer and put the drawer in place.
6. Insert the PDQeX cartridges into the heating block.
7. Cover the cartridges with the hinged flap and close thesliding door.
MAKE SURE THE PDQeX CARTRIDGES CORRESPOND WITH A
COLLECTION TUBE OR WELL - OTHERWISE YOU WILL LOSE YOURSAMPLE

8. Select the “Plant” program.
52 ˚C
75 ˚C
95 ˚C••

5 min
5 min
2 min

Times may be adjusted during method optimisation.
Changes to the default temperatures are not recommended.

Important Technical tips•

The device must be kept clean and DNA-free. The UV cycle willassist in removing DNA but the wells and the top and base of theheating block should be cleaned regularly using dilute HClO
bleach on a swab. Wipe residual bleach off the surfaces using asecond swab soaked with DNA-free water.

•The device produces DNA that can be used for SNiPs, STRs, quantitative, multiplex and routine PCR applications.

•OD260 methods for yield estimation are generally unsuitable forthe DNA produced by the PDQeX. For accurate yield assessment,a qPCR or fluorometric assay is recommended.

•For best results prepare and manage samples at 4º C, or on ice
before and after extraction.

•For long term storage of the extracted DNA, add TE buffer to 1x
and store at -20° C.

•

The phytoGEM extraction chemistry and cartridges are designed
to remove polyphenols, polysaccharides and other inhibitory
compounds from the extract. Nonetheless, some plant samples
will have more inhibitory material than others - especially iflarge amounts of extract are used in the PCR. The following
suggestions should be tried if you experience any problems
with your PCR:
1. Make sure that the problem is inhibition and not a low
concentration of DNA. Inhibition can be tested with a control
where one of your extracts is spiked with a known quantity ofDNA. Alternatively, if you are using qPCR, the slope and endpoint of the plot gives a good indication of inhibition.
2. Add the Enhancer to the PCR mastermix. Adding 2.5 µl of theEnhancer in a 25 µl PCR may improve results from plants withhigh polyphenolic content.
3. Add BSA to the PCR mastermix. 1 µl of a 10 mg/ml solution ofBSA in a 25 µl reaction is sufficient for reducing the effects ofcommon PCR inhibitors.
4. Try adding less sample to your PCR. Usually 1 µl or less of theextract is enough for a successful PCR. It is good to optimise
the amount of extract added for every new sample type youtest.