Download DNAready Lysis Buffer

DNAready Lysis Buffer
to extract the DNA from bacterial suspensions as a previous step before PCR
DNAready is a new generation lysis buffer; specifically developed to quickly extract DNA from bacterial suspensions (like enrichments in Half
Fraser broth or Buffered Peptone Water) allowing further amplification by real time PCR. It could be also used to extract the bacterial DNA
from water environmental samples after its filtration. DNA ready is the ideal complement for Microbial SL detection systems (Salmofast®,
Listerfast®, Campylofast THERMO, Cronofast, Bactoplex, Bactoplex PIF, Legiofast® ENVIRON and Legiofast® SPECIES). The recommended protocol for
DNAready has been optimized to obtain a better yield with fewer steps, in both gram-negative and gram-positive bacteria. Thus, most of the
PCR inhibitors that could be present in bacterial suspensions are removed without performing laborious and time-consuming silica-gel
column purification steps.
PROTOCOL
1. From bacterial cultures/enrichments:
Spin 1 ml of the sample enrichment for 5 minutes at 8000 g. Discard the supernatant.
When using Bactoplex, spin 0.9 ml of Salmonella spp. enrichment and 0.9 ml of Listeria monocytogenes enrichment in the same tube.
When using Cronofast or Bactoplex PIF, it is possible to mix directly 100 µl of the enrichment with 200 µl of DNAready Lysis Buffer, homogenize pipetting or
vortexing and move to the step 3 without spinning.
2.
3.
4.
5.
6.
From environmental samples (water):
Filter the volume of water to be analyzed with 0.45 µm membranes (polycarbonate or another compound capable of DNA and protein
retention).
Recover the filter or the surface adhered particles aseptically (with a handle or spatula) and introduce it into a microcentrifuge tube.
Resuspend the pellet or the filter with 200 µl of DNAready Lysis Buffer. Mix pipetting or vortexing until the complete dissolution of the
pellet or the filter adhered particles.
In some cases, a greater volume of DNAready Lysis Buffer may be needed in order to obtain a complete dissolution of the filter.
Incubate 30 minutes at 56ºC.
Incubate 10 minutes at 95ºC.
Incubate 5 minutes at -20ºC.
Proceed with the real time PCR with the required Microbial kit. Add 1 µl of the resultant extracted DNA into the corresponding tubes or
plate wells with 19 µl of Reaction Mix. DNA ready inactivates most of the PCR inhibitors allowing a correct amplification reaction. In
some samples, like enrichments in Half Fraser to detect Listeria monocytogenes, it is strongly recommended to dilute the sample 10 or
100-fold before the real time PCR.
* It is not recommended to mix the final DNA extract from enrichments with a high content of particles in suspension to avoid the
resuspension of materials which could inhibit of the amplification reaction.
Bacterial cultures/Enrichments
Environmental samples (water)
Filter the sample
1 ml enrichment
Spin 5min
at 8.000 g
Introduce the filter
into the microtube
Discard supernatant
Resuspend with 200 µl of
DNAready Lysis Buffer
Resuspend with 200 µl of
DNAready Lysis Buffer
Incubate:
30 min 56ºC
10 min 95ºC
5 min -20ºC
Incubate:
30 min 56ºC
10 min 95ºC
5 min -20ºC
Proceed with 1 µl to real time
PCR (dilute 1/10 or 1/100 if
necessary)
Proceed with 1 µl to real time
PCR (dilute 1/10 or 1/100 if
necessary)
Note: DNAready has been satisfactorily tested in bacterial enrichments of many food samples, like dairy products, eggs, raw vegetables, fruits, fruit juices,
precooked dishes, raw and cured meat, poultry, raw and smoked fish, chocolate and ice-creams. However, some food samples may be problematic. If
amplification of neither the detector nor the internal amplification control is detected, even if diluting the sample, repeat the DNA extraction with a silica-gel
column purification method. DNAready is not recommended to extract DNA from bacteria without performing a previous enrichment step.
RECOMMENDATIONS
The use of filter stomacher bags may help to avoid, or at least reduce, the presence of inhibitory particles in case of enrichments with great
abundance of suspension particles.
Food matrices with a high fat content or with antimicrobial agents are often problematic since they interfere in the enrichment of the bacteria
to detect. In these situations it could be useful to increase the growing time, perform a second selective enrichment or, in the specific case of
high-fat samples, carry out the enrichment with Tween 80 at 10%.
STORAGE
Store at 4ºC or at -20ºC. Avoid frequent freeze/thaw cycles.
Microbial SL - PCiT UdG, Edifici J Casademont E - Pic de Peguera 15 - 17003 Girona (Spain) phone: +34 972 183 236