Vivantis Bacterial DNA extraction kit

[magicsiew]

I am using Vivantis Bacterial DNA extraction kit to extract DNA from Achantameoba culture. I follow exactly the protocol provided along with the kit. However, the amount of DNA that I able to able to extract is very minute, with concentration just few ng/ul when measure by NanoQuant. No band formed from AGE. I repeat twice and get the same result. Anyone got any idea what is the problem?

[canalon]

I am neither familiar with the vivantis kit nor with achantamobea, but often those kits are tested with common lab cultures of a few Gram + and - and may not work as well with other species (or even environmental isolates). Usually because the cell wall is different is different and not properly degraded.
In your kit they seem to rely on proteinase K, just like Qiagen, and in my experience it is far form good for even E. coli environmental isolates.
There are alternative technologies to lyse the cells: bead beating (great for everything, but lots of shearing), boiling or even autoclaving, other chemicals...
Maybe you could have a look at the literature for your organism to see what people usually use.

[magicsiew]

Hi there, yes I have search for some literature and found there this Acanthamoeba need some pre-treatment before the commercial kits. By the way, how do you mean by autoclaving to lyse the cell? Usually in which step we can do autoclave?

[canalon]

I mean that:
http://www.ncbi.nlm.nih.gov/pubmed/14744443
I have not tried, because at the time it could have been interesting I could not access to the programs of the autoclave, and now, it is of no use to me...

[JackBean]

I thought, that autoclaving destroys the DNA...

[canalon]

It does after a while, in this case, the time spent at high temperature is very limited enough to cause the bacteria to lyse, but not enough to destroy DNA. That is one reason you need to have the rights to program your autoclave to use this method, since your usual programs are quite useless.

[JackBean]

so it's basically the same as when you use bacteria directly for PCR...

[canalon]

Yes and No. Because many bacteria will not break open with just boiling.
Direct colony PCR is nice for your average E. coli but will fail with many natural isolates with capsules. And this is also very true with other groups such as Gram positive that are in general much harder to lyse with simple boiling. Autoclave on the other hand is supposed to be universally efficicent.

[magicsiew]

I have take a look at my lab autoclave machine.. I think I cant change the setting, as just got the Start and Stop button along with the meters only. However, i do found some can change the setting, just that not yet getting the approval...

[canalon]

Let us know if it works well.

[magicsiew]

Not sure if the lab officer allow me to change the setting or not yet. If allow to do so, will let you all know.