MicroGEM Quick-Start Guide

DNA Extraction Using

PDQeX
Bacteria

MicroGEM
Going to extremes for
effortless DNA extraction

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QSG_006_190531_PDQeX prepGEM Bacteria

About this Guide

MicroGEM

This Quickstart guide provides a number of efficient methods to suit a variety of bacterial
morphologies and substrate types. These methods can be further optimised and adapted to suit
your sample type. We are available to provide help with developing a custom method. Pleasecontact us at info@microgembio.com

Preparation and Storage of Reagents

MicroGEM•Lysozyme is provided as a lyophilised powder. To use, resuspend in 100 mM Tris pH 8.0 to
the volume specified on the label. Some of the powder might dislodge from bottom of thetube during shipping. It is recommended to spin this down before resuspending.

•To reduce the potential for contamination and activity loss, the Lysozyme and prepGEM are
best stored in small aliquots at -20°C.

•The WASH+ buffer is provided at 5x concentration. This needs to be diluted to 1x using
DNAase-free water prior to use in protocols. Aliquots of 1x stocks can be made and stored
at room temperature or at 4˚C for long-term storage.

•GREEN+ Buffer and Enhancer need to be stored at 4˚C.

Precautions

MicroGEM

1.

Do not load the PDQeX machine if the control screen indicates a temperature above 35° C.

2.

Ensure the collection drawer and heating block are clean and DNA-free.

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 used in the heating block.

MicroGEM Reagent QC

MicroGEM

Microbial DNA in reagents is a well-known problem for microbiologists. MicroGEM goes to great
lengths to minimise this problem. Our reagents are made from certified DNA-free chemicals andsolutions and all buffers and enzymes are treated with DNAse and UV before shipment. Be aware
however, that we have no control over the reagents of other vendors. If you are using universal
primers in a PCR (for example 16S rRNA gene primers) you should look at published literature
about how to reduce the background signal you may get from your PCR or qPCR reagents.

Removing Inhibitory Polysaccharides and Polyphenols

MicroGEM

PDQeX Bacteria comes with WASH+ buffer. Some samples require a pre-wash step. Typically,
these samples are:••••

Bacteria producing large amounts of polysaccharide
Capsulated bacteria
Samples presented in mucous (for example sputum, throat or vaginal swabs)
Samples in a matrix of inhibitory tannins, humic substances and polysaccharides. Forexample soil and stool.

The WASH+ buffer is a proprietary formulation designed to reduce these problems. It is a blendthat binds inhibitors, breaks down mucus and is osmotically buffered. Osmotic buffering is
critical when mixed samples are presented composed of fragile and robust bacteria.

Important Technical Tips

MicroGEM•DNA extracted using the PDQeX Bacteria kit is suitable for Whole-Genome Sequencing andmany types of genotyping including SNP and STR analysis as well as quantitative, multiplex
and end-point PCR.

•The amount of starting material to use in extractions depends on the sample type. It is recommended that you optimise this by testing different amounts of your specific sample. For
PCR-based applications, a tiny amount would suffice. For applications requiring high DNA
yields like Whole-genome sequencing, ~2 mm² of colony or 20 µl of log-phase liquid culture
is sufficient for most samples.

•The methods provided can be further optimised for different samples. These methods aredesigned to limit the effect of inhibitors. If inhibition remains a problem, adding 2.5 µl of
the Enhancer to a 25 µl PCR mastermix might improve results, especially for stool and soil
samples. Try different amounts of extract in your PCR - sometimes less is better. Using 1 µlor less in 25 µl PCRs is often sufficient for bacterial amplifications.

•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 PDQeX Bacteria can be quantified using a qPCR or by using fluorescentdyes like Pico Green, iQuant, Qubit assays or the like. Nanodrop is incompatible with PDQeXreagents.

•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.

Colonies, Biofilms, Liquid Cultures, Sputum and Swabs

MicroGEM

Completely thaw prepGEM and Lysozyme, and mix by gently inverting the tubes. Remove
GREEN+ and WASH+ buffers from the refrigerator and mix.

Colonies and Biofilms
Cells from colonies can be suspended directly into the extraction mixture. Do not be tempted topick up too much of the colony, it might clog the PDQeX cartridge (Refer to ‘Important technicaltips’ section for recommendations on amount of sample to use).
A pre-wash in WASH+ buffer is recommended for biofilms and high exo-polysaccharide
producers.
1.

Pipette 400 µl of 1x WASH+ buffer into a 1.5ml Eppendorf tube.

2.

Lift a small amount of colony with a sterile loop or pipette tip or a biofilm and resuspend
in WASH+ buffer.

3.

Vortex vigorously to disperse cells.

4.

Centrifuge the cells at >10,000 r.c.f. for 5 minutes.

5.

Remove ALL of the supernatant and discard.

6.

Resuspend the pellet in the extraction mix below:

Liquid Cultures and Sputum
The amount of culture to add is dependent on the density (Refer to ‘Important technical tips’
section for recommendations on amount of sample to use)
1.

Pipette up to 20 µl of log-phase culture or 20-100 µl of sputum in a 1.5 ml Eppendorf
tube.

2.

Add 400 µl of 1x WASH+ buffer.

3.

Vortex vigorously to disperse cells.

4.

Centrifuge the cells at >10,000 r.c.f. for 5 minutes.

5.

Remove ALL of the supernatant and discard.

6.

Resuspend the pellet in the extraction mix below:

Swabs
1.

Wash swab for 30 sec in 400 µl of 1x WASH+ buffer in a 1.5ml Eppendorf tube using
a rolling action. Before discarding, squeeze the swab head on the wall of the tube toextract as much of the liquid as possible.

2.

Vortex vigorously to disperse cells.

3.

Sediment the cells by centrifugation at >10,000 r.c.f. for 5 minutes.

4.

Remove ALL of the supernatant and discard.

5.

Resuspend the pellet in the extraction mix below:Preparing the extraction mixture
For each extraction, make up:
86 μl

DNA-free water

10 μl

10x GREEN+ Buffer

2 μl  	 prepGEM
2 μl

Lysozyme (Can be omitted for Gram negatives)

PDQeX extraction
1.

Dispense 100 µl of the extraction mix with sample into each PDQeX cartridge.

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 appropriate program below for you sample:
Gram Neg Bacteria:
75°C

10 mins

95°C

2 mins37˚C

10 mins

75°C

10 mins

95°C

2 minsGram Pos Bacteria:•Times may be adjusted by internal laboratory optimisation.

•Changes to the default temperatures are not recommended.

Soil and Stool

MicroGEM

Completely thaw prepGEM and Lysozyme, and mix by gently inverting the tubes. Remove
GREEN+ and WASH+ buffers from the refrigerator and mix.
Extracting DNA from soil samples for PCR and qPCR is complicated because of the release ofhumics. This method uses a short differential sedimentation of solids in the WASH+ buffer. Thisbuffer is designed to dislodge bacteria from biofilms but it is isotonic to protect more fragile
species.

Differential sedimentation step1.

Add up to 10 mg stool or up to 20 mg soil to a 1.5 ml tubes (this can be optimised foryour sample).

2.

Resuspend in 500 μl of 1x WASH+ buffer.

3.

Vortex vigorously for 1 min to disperse cells.

4.

Centrifuge gently at 200 r.c.f for 30 seconds to sediment the solids but leave asuspension of cells.

5.

Transfer the supernatant to a new tube.

6.

Centrifuge at full speed for 2 min.

7.

Carefully pipette away all of the WASH+ buffer.

8.

Resuspend pellet in 100 μl water.

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

10x GREEN+ Buffer

10 µl

Enhancer

2 μl

Lysozyme (10 mg / ml)

2 μl

prepGEM

76 μl

Water

50 μl

Cell suspension

PDQeX extraction
1.

Dispense the above extraction mix with sample into each PDQeX cartridge.

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 SAMPLE7.

Select the appropriate program for you sample (below):
Soil and Stool:
37˚C

15 mins

75°C

15 mins

95°C

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

•Changes to the default temperatures are not recommended.