MicroGEM PDQeX prepGEM Universal Effortlessly Extract DNA From Saliva or Blood User Guide

Quick Start – Guide: PDQeX prepGEM Universal

This Quick Start Guide provides methods to extract DNA from a range of sample types.

Principle of the Extraction

MicroGEM extraction products use a unique mixture of thermophilic and mesophilic enzymes. Protocols involve a 75°C step to activate MicroGEM’s thermophilic proteinase to lyse cells, denature nucleases, and strip DNA of nucleosomes. Certain protocols in this Quick Start Guide include an initial low-temperature step when mesophilic enzymes are required. A final 95°C step shrinks the PDQeX inner tubing, bursting the valve and pushing the extract through a column at the base of the tube to remove the proteinase and inhibitors, resulting in analytics-ready DNA being released from the tube.

Kit Components and Storage Conditions

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

• Bacteria produce 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. For example soil and stool.

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

• PDQeX prepGEM bacteria reagents are delivered at room temperature but on arrival should be stored at 4˚C. After tubes have been opened, the prepGEM enzyme should be placed at -20˚C.
• Lysozyme is provided as a lyophilized powder, to use, resuspend in 100 mM Tris pH 8.0 to the volume specified on the label. Once lysozyme has been rehydrated, it is stable for several weeks at 4˚C. If you do not plan to use all of the Lysozyme immediately, it is recommended that you aliquot lysozyme into smaller volumes and store at -20˚C immediately after rehydration. The GREEN+ buffer can remain at 4˚C for convenience.
• The WASH+ buffer is provided at 5x concentration and may appear yellow. This needs to be diluted to 1x using nuclease-free water prior to use in protocols. You may find that the 5x WASH+ buffer contains some floating crystals, you should warm the 5x WASH+ buffer to ~35˚C to dissolve these fully before diluting to 1x. Aliquots of 1x stocks can be made and stored at room temperature or at 4˚C for long-term storage.

MicroGEM Reagent QC

Microbial DNA in reagents is a well-known problem for microbiologists. MicroGEM goes to great lengths to minimize this problem. Our reagents are made from certified DNA-free chemicals and solutions 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.

General Instructions, Precautions, and Technical Tips

• DNA extracted using the PDQeX prepGEM Universal kit is suitable for Whole-Genome Sequencing and many types of genotyping including SNP and STR analysis as well as a 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 optimize 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 the colony or 20 pl of log-phase liquid culture is sufficient for most samples.

• The methods provided can be further optimized for different samples. These methods are designed to limit the effect of inhibitors. If inhibition remains a problem, adding 2.5 pl of the Enhancer to a 25 pl PCR master mix might improve results, especially for stool and soil samples. Try different amounts of extract in your PCR – sometimes less is better. Using 1 pl or less in 25 pl PCRs is often sufficient for bacterial amplification.

• There is no concentration step in the procedure and so the concentration of the extract is depen­dent on 1) The quality of the sample; 2) In the case of swabs, the type of swab and the volume of water used to wash the swab; 3) The extraction volume (which in some cases can be scaled).

• DNA extracted using PDQeX prepGEM Universal can be quantified using a qPCR or by using fluorescent dyes like Pico Green, piquant, Qubit assays, or the like. Nanodrop is incompatible with PDQeX reagents.

• As with any preparative method for nucleic acid extraction, the 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 lx (10 mM Tris, pH 7.5, 1 mM EDTA) and store at -20°C.

Sample Preparation and Extraction Procedures

Colonies, Biofilms, Liquid Cultures, Sputum and Swabs
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 to pick up too much of the colony, it might clog the PDQeX cartridge (Refer to
‘Important technical tips’ section for recommendations on the amount of sample to use). A pre-wash in WASH+ buffer is recommended for biofilms and high exo- polysaccharide producers.

1. Pipette 400 pl of lx 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 x g for 5
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 the amount of sample to use).

1. Pipette up to 20 pl of log-phase culture or 20-100 pl of sputum in a 1.5 ml Eppendorf tube.
2. Add 400 pl of lx WASH+
3. Vortex vigorously to disperse cells.
4. Centrifuge the cells at >10,000 x g 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 1.5 ml Eppendorf tube using a rolling action. Before discarding, squeeze the swab head on the wall of the tube to extract 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, makeup:

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 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 appropriate program below for your sample:
   Gram Neg Bacteria: 75°C| 10 mins
   ---|---
   95°C| 2 mins

Gram Pos Bacteria:

Soil and Sperm
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 of humic. This method uses short differential sedimentation of solids in the WASH+ buffer. This buffer is designed to dislodge bacteria from biofilms but it is isotonic to protect more fragile species.

Differential and Sedimentation Step

1. Add up to 10 mg stool or up to 20 mg soil to 1.5ml tubes (this can be optimized for your sample).
2. Resuspend in 500 μl of 1x WASH+ buffer.
3. Vortex vigorously for 1 min to disperse cells.
4. Centrifuge gently at 200 x g for 30 seconds to sediment the solids but leave a suspension 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+ buffers.
8. Resuspend pellet in 100 μl water.

Preparing Extraction Mixture

For each extraction, makeup:

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 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 appropriate program for your sample (below):
   Soil and Stool:  37˚C| 15 mins
   ---|---
    75°C| 15 mins
    95°C| 2 mins

Troubleshooting

For troubleshooting, please see our guide “MicroGEM Method Optimization and Troubleshooting” using the following link:
https://www.microgembio.com/m_resource/microgem-method-optimisation-and- troubleshooting/

Additional Information

For publications/app notes on your specific sample type, please visit: https://www.microgembio.com/resources/

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At MicroGEM, our goal is to democratize molecular biology, enabling a broader spectrum of users to both employ and benefit from molecular techniques. The first step is the simplification of sample preparation. Our temperature- driven, single-tube process simplifies and reduces the number of steps for traditional nucleic acid extraction, resulting in high-quality extracts with reduced contamination and high yields – all in minutes, not hours.

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