MicroGEM Quick-Start Guide

DNA Extraction Using
prepGEM Bacteria

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_002_190531_prepGEM Bacteria

Bacteria
This quick start guide provides a number of efficient methods tosuit a variety of bacterial morphologies and substrate types. Thesemethods can be further optimised and adapted to suit your sampletype. We are available to provide help with developing a custom
method. Please contact us at info@microgembio.com.
We recommend that you visit: www.microgembio.com for more
information.
QC of MicroGEM reagents
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-freechemicals and solutions and all buffers and enzymes are treatedwith DNAse and UV before shipment. Be aware however, that
we have no control over the reagents of other vendors. If you areusing universal primers in a PCR (for example 16S rRNA geneprimers) you should look at the literature about how to reduce thebackground signal you may get from your PCR reagents.
Preparation of the Reagents•Lysozyme is provided as a lyophilised powder. To use,
resuspend in 100 mM Tris pH 8.0 to the volume specified onthe label.

•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 needsto be diluted to 1x using DNAase-free water prior to use inprotocols. Aliquots of 1x stocks can be made and stored atroom temperature or at 4˚C for long-term storage.

Bacterial method overview

Colonies and Biofilms
Removal of Inhibitors
The WASH+ buffer is a proprietary formulation designed to reduce
problems caused by inhibitory polysaccharides and polyphenols
in the sample. A pre-wash in this buffer is recommended for:•bacteria producing large amounts of polysaccharide•

capsulated bacteria•

samples presented in mucous (for example sputum, throator vaginal swabs)•

removal of inhibitory tannins, humic substances andpolysaccharides from soil and stool
Proceed with a method outlined below

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. A
pre-wash in WASH+ buffer is recommended for biofilms and highexo-polysaccharide producers.
1.

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

2.

Lift a small amount of colony with a sterile loop or pipettetip (up to 2 mm2) 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, Sputum and Swabs
Liquid Cultures and Sputum
The amount of culture to add is dependent on thedensity. Using 20 µl of log-phase culture yields
good results. This can be optimised for yoursample.
1.

Pipette 20 µl of log-phase culture or 20-100µl 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 5minutes.

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 1x WASH+buffer in a 1.5 ml Eppendorf tube using arolling action. Squeeze the swab headagainst the wall of the tube to extract asmuch liquid as possible before discarding.

2.

Vortex vigorously to disperse cells.

3.

Centrifuge the cells 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:

Extraction
Extraction Mixture
For each extraction, make up:		88 µl		10 µl		 1 µl		 1 µl

DNA-free water
10x GREEN+ Buffer
prepGEM
Lysozyme (Omit for Gram -ves)

Extraction Incubations
Incubate at:
37°C for 15 min (Omit for Gram -ves)
75°C for 10 min
95°C for 2 min
A thermal cycler can be used for this procedure

THE DNA IS NOW READY FOR USE

Soil and Stool
Extracting DNA from soil samples is complicated due to the releaseof humics. This method relies on a short differential sedimentationof solids in a proprietary buffer.
Differential Sedimentation Step1.

Add up to 50 mg of soil or stool to a 1.5 ml tube.

2.

Resuspend in 500 μl of 1x WASH+ buffer.

3.

Vortex vigorously for 1 min to disperse cells.

4.

Centrifuge at 200 rcf for 30 seconds.

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 of water.

Extraction
1.

Make up the following extraction cocktail:		78 µl		10 µl		 10 µl		 1 µl		 1 µl		50 µl

2.

DNA-free water
10x GREEN+ Buffer
Enhancer
prepGEM
Lysozyme
Cell suspension

Incubate at:
37°C for 15 min
75°C for 10 min
95°C for 2 min

3.

Centrifuge at full speed for 2 min.

The supernatant contains the DNA. Use 1 µl or less for PCR/qPCR

Rapid plasmid prep from E. coli
1.

Grow overnight cultures to an OD600 of approximately1-1.5.

2.

Transfer 200 µl of the culture to a thin-walled 200 µl PCR
tube.

3.

Centrifuge at full speed for 2 minutes and pipette away themedium from the pellet.

For colonies, scrape a colony of approximately 4 mm diameterand use in the extraction below.
Extraction
1.

Add to the pellet and mix:
5 µl
44 µl
1 µl

2.

10x GREEN+ Buffer
DNA-free water
prepGEMIn a thermal cycler, incubate at:
75°C for 5 min
99°C for 5 min
4°C for 2 min (Rapid cooling step should be included)

3.

Centrifuge for 5 minutes at 20,000 r.c.f.

4.

Discard the pellet. The supernatant contains the plasmid.

Typical results for high copy
number plasmids extractedusing prepGEM Bacteria

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.
The prepGEM 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 at www.microgembio.com