PSP Spin Stool DNA Kit - STRATEC Biomedical AG

More documents

Recommendations

Info

A260/A280 ratio for purified nucleic acids islowWash Buffer I and Wash Buffer II used in thewrong orderprotein contaminationensure that Wash Buffer I and Wash Buffer II are used inthe correct order in the protocol.repeat washing step with Wash Buffer I in the repeatedpreparationDNA does not perform well in downstreamapplicationsBSA not added to PCR mixturetoo much DNA used in downstream reactionnonspecific bands ininefficient lysis of target cellsnot enough DNA in eluateinhibitory substances in preparationwhen using eluates in PCR, for maximum PCR robustnessadd BSA or I-Solution to a final concentration of 0.1 μg/μl tothe PCR mixture.the PSP ® Spin Stool DNA Kit and PSP ® Spin Stool DNAPlus Kit purifies total DNA, which could originate from manydifferent organisms present in the original stool sample (e.g.,human, animal, plant, bacterial). If the amount of total DNAis too high, PCR could be inhibited by excess total DNA.Reduce the amount of eluate used in the downstreamreaction if possibleit is likely that only a low quantity of target downstream PCRDNA is present in stool-sample eluates, together with highamounts of background DNA. To maximize PCR specificity,it is recommend the use of InviTaq Hot Start Taq DNAPolymerase (see ordering information on page 30).the amount of target DNA in the eluate may be low if thetarget cells are difficult to lyse, as is the case with somebacteria and parasites. In future preparations, prolongincubation time of the sample at 95°C and/oradd zirconia beads to the stool samples lysis mixture (seePSP ® Spin Stool DNA Kit protocol 2, page 17.)check “Low amount or no DNA of extracted DNA” forpossible reasons.see “A260/A280 ratio for purified nucleic acids is low” forpossible reasons. Bring the eluate volume to 200 μl .add tothe supernatant 400 µl Lysis Buffer P and mix all with 200µl Binding Buffer A. Repeat the protocol 1 from step 5 of“Isolation of total DNA from up to 400 mg stool samples withand without enrichment of bacterial DNA “ (page 16).See also protocol: Post Purification, see page 25residual Wash Buffer in the eluateensure that the Wash Buffer I and II are used in the correctorder in the protocol.add 400 µl Lysis Buffer P and 200 µl Binding Buffer Ato the eluate, and continue with step 5 of“Protocol: Isolation of total DNA from up to 400 mg stoolsamples with and without enrichment of bacterial DNA “(page 15.PSP ® Spin Stool DNA Kit 0213PSP ® Spin Stool DNA Plus Kit 0213
insufficient mixing with Lysis Buffer P or StoolDNA Stabilizerreduced sensitivity of amplification reactionrepeat the purification with other aliquotsdetermine the maximum volume of eluate amplificationreaction suitable for your amplification reaction. Reduce orincrease the volume of eluate added to the reactionoptimize your amplification system e.g. by changingtemplate volumeLittle or no supernatant visible after initialcentrifugation stepinsufficient centrifugal forceLittle or no supernatant visible aftercentrifugation step with InviAdsorb matrixinsufficient centrifugal forceincrease the centrifugation time proportionately if yourcentrifuge cannot provide 13.400 x g (12.000 rpm), e.g.instead of centrifuging for 1 minutes at 13.400 x g, centrifugefor 3 minutes at 10,000 x g).with some samples, centrifugation to precipitate theInviAsorb matrix may result in a pellet that is not sufficientlycompact.in these cases, it is recommended to increase thecentrifugation time for precipitation of InviAdsorb matrix to6 minutes.Precipitate after addition of Binding Buffer An most cases, this effect comes frm big amounts of DNA inthe sample. Don’t remove this precipitate and follow strictlythe protocol.General handlinglysate not completely passed through silicamembranecross-contamination between samplescentrifuge for 1 minute at full speed or until all the lysate haspassed through the membrane.to avoid cross-contamination when handling RTA Spincolumns, read “Handling of RTA Spin Filter “ on page 12.Repeat the purification procedure with new samples.PSP ® Spin Stool DNA Kit 0213PSP ® Spin Stool DNA Plus Kit 0213
Page 8 and 9: Intended useThe PSP ® Spin Stool D
Page 10 and 11: Product characteristic of the PSP
Page 13 and 14: ProcedureLysisStool samples are lys
Page 15 and 16: 5 total DNA extractions:add 250 µl
Page 17 and 18: Scheme of the PSP ® Spin Stool DNA
Page 19 and 20: 3. Second sample cleanupTransfer th
Page 21 and 22: 5. Binding of the DNAAdd 200 µl of
Page 23 and 24: InstructionsThe following notes are
Page 25 and 26: 5. Binding of the DNAAdd 400 µl of
Page 27 and 28: 6. Washing StepsPut the RTA Spin Fi
Page 29: TroubleshootingProblemComments and
Page 33 and 34: Ordering informationProduct Package

PSP Spin Stool DNA Kit - STRATEC Biomedical AG

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?