DNA extraction has been done by Quiagen Stool Mini kit, ASL buffer is used. Stool Sample were properly stored at -20. Extracted DNA were stored at -80, no freeze thaw and proper protein precipitation was done.
Just to run on 1% agarose gel to check the inegrity of you metagenomic DNA along with do a 16s specific PCR (region would be V1-V4 in between) and concentration should be checked with Qubit before library prep.
We have done all this already and there is DNA degradation!
How to (check), I mean prevent it!
ok I got you, first of all did you get PCR amplification from that metagenomic DNA ??
if yes then what is your initial DNA concentration by Qubit or Nanodrop ?
Few thing which I was expericed from fecal DNA extraction is that metagenomic DNA concentration is quite low, though I initially got degraded DNA but then optimized it at the initial storage temperature and by through proper handling.
Out of 6 metagenomic DNA samples we extracted, we got 16S amplification in only one sample (initially all 6 were 16S positive). We ran it again in gel and although DNA is visible some smearing is there. Our DNA concentration was very good, >50 ng per microl, as measured by nanodrop. Infact we were also getting low DNA initially but we discovered that we can get good level when we elute again! Today we are doing PCR on 2nd aliquot of our sample which was stored at -20 C.
We have kept the samples at -20C. Sample aliquot were taken n processed on ice We are not following bead beating process. After DNA extraction, we checked it by gel visualization than we quantified it by nanodrop and we got good quantity (as mentioned above). 3 aliquots were obtained and first one was kept at -80 and 2 at -20. We are checking stored samples by 16S PCR. Thanks for your thoughts and yes we will again go step by step as u said!
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