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firefly2280

Member Since 05 Mar 2012
Offline Last Active Jan 19 2013 10:39 AM

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My research interests
molecular biology, environmental sampling, POC diagnostics, Viral diseases, ebola etc..!

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Topics I've Started

Making a standard curve from bacterial suspensions

19 January 2013 - 10:37 AM

Hi

Sorry if this is an obvious question but Ive just been confusing myself!

I want to make a standard curve for S. aureus to run on my qPCR. I have bacterial suspensions - say 10^8 / ml.

If I just do a bead-beat and lyse the cells, proteinase K treat then etc, then I have a suspension that I can say is 10^8.

But then if I add 10ul to a qPCR, im only actually adding 10^6 gene copies, right? So if I want to do it this way, I need to start at a much higher dilution?

Is this right or am I totally off the mark?

Then, if I get 10ul of sample come up at 10^6, does that mean I have 10^6 in my 10ul of sample!?

thanks so much

Best method for extraction on low concentration environmental DNA?

05 November 2012 - 08:43 AM

Hi all

I could really do with some help. I am swabbing environmental sites, ie floors, surfaces (a 10cm3 area) and trying to extract DNA. Needless to say, Im not having a lot of luck, I think quantities are very low and inhibitors are present.

I have tried Qiagen colums with a bead-beating step first and also Phenol:chloroform. The column based method was better but neither give me a lot of DNA.

Does anyone have any ideas about increasing my yield please?

thanks

Reference for crude bacterial DNA prep?

09 July 2012 - 05:32 AM

Hi

Ive been boiling and bead-beating pure cultures to extract DNA and now writing up need a reference for it...! Does anyone have one to hand please?

Thanks