I would like to carry out qPCR for quantification of Salmonella and E.coli from pork samples and vegetable salads.
I would like to extract and store the bacterial cells first as pellets before proceeding with DNA extraction after a few months.
I can't seem to find a protocol online for bacterial cell extraction.
I would appreciate any leads.
If I got your point correctly, you can isolate your bacteria from your samples and then freeze cells without any buffer for DNA, but if you are going to check expression you have to use RNAlater buffer.
After growing the bacterial cells in a broth, harvest by microcentrifugation or ultracentrifugation. You can wash the cell pellets with phosphate buffer saline or other buffer solutions. Since you are thinking of storing the pellets for a long time, I will suggest you store them in a cryoprotectant such as glycerol. This will help in preventing the membrane distortion caused by freeze-thawing cycle.
All the best,
Thank you Alireza Mordadi and Ifeanyichukwu Eke .
I plan to harvest the bacterial cells directly from enriched food and fecal samples. The plan is to collect the samples, enrich in buffered peptone water for 18 hours and then proceed to harvest them. Does the protocol remain the same i.e. microcentrifugation? and how much will I need to centrifuge so that I get enough for quantification (cfu/g) by quantitative-PCR?
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