4 replies to this topic

[joy123]

Hi, I want to check microbe 16srRNA from human sample (liquid). I want to know if it is propriate to store the samples (liquid) at -80C for months, and extract DNA later?

Are there any special needs? For instance, adding agents to stabilize DNA?

Thanks a lot!

[bob1]

It should be fine to store at -80, RNA is pretty stable at that temperature, and if it is wrapped up in the ribosome, it is even more stable.

[phage434]

And you probably will really extract DNA rather than RNA, and then amplify the 16s rRNA fragment of the genomic DNA.

[dick123]

I think yes i agree with you.

[joy123]

Hi! Thanks a lot for your responses. I have just got the sample.

1. which general procedures should I go through? I think it includes: DNA extraction-->get primers for the specific 16srRNA--> do common PCR to check if the primers are good-->do RT-PCR (include positive and negative control)-->data analysis. Is that correct?

2. The sample contains mammal cells and bacteria (gram- and grm+). I am interested in a G+ bacteria 16srRNA. As far as I know, the G+ bacteria DNA are harder to extract. How should I extract the DNA?

3. How should I design the primers for 16srRNA? Which database is good for designing the specific primer?

4. If the extracted DNA contains mammal cells DNA, and bacteria DNA, how should I quantify the DNA to make sure I load the same amount of bacteria DNA?

Thanks a lot! Look forward to your responses!