QCMD samples - (Sep/26/2010 )
Any body using QCMD (Quality control for molecular Diagnostics, Glasgow) samples for HBV genotyping?
Few days back I received the serum samples. Extracted the DNA and had setup the PCR, however when I had setup the PCR for the second time no amplification took place. I tired it many times by extracting the DNA again but no amplification. This has not happened the 1st time. A year ago same happened like after setting up the PCR for about 1st or the second time. The DNA stopped working.May be the DNA gets degraded or I don't know. Weird..... Any one knows the answer? DNA is extracted by using Qiagen DNA blood mini kit.
how do you store the extracted dna (buffer, temperature)?
if you store in water then your dna may be degrading.
Well after extraction and for further usage I store DNA @ -70C and this is what is surprising that how does it
gets degraded at this much low temperature.
you could be fracturing the dna with ice crystals.
but, the damage may be occurring when the sample is defrosted. if it is in unbuffered water then you may be acid hydrolyzing the dna.
Well this is not only done with the QCMD samples.. I have extracted and stored a lot of viral DNA using the above mentioned protocol and conditions ( I have got about 2 years old viral DNA which is working with my PCR's) but with these QCMD samples some mystery is there and what comes to my understanding is that there is something in the serum added by the company (QCMD)which degrades the DNA after its first usage. The reason being that these samples are expensive and are HBV type specific (A-G) in each vial which is of great value for most of the diagnostic labs in order to optimize there HBV genotyping PCR assay which without them (QCMD samples) is very difficult (nearly impossible).... so may be this is the reason .... or is there anyone out there with some suggestions to this matter??
and the DNA eluted in the AE buffer (Qiagen)