7 replies to this topic

[303microbialist]

Hi all,
I'm hoping I can get a bit of advice on some issues I'm having with my current project:

I use "universal" bacterial 16s primers, 8F/1392 in this case, to amplify environmental soil DNA extracts for use to clone and sequence.

We found a seemingly biotic crust (bright green and yellow on a white hard base) at around 18,000 ft on a volcano in South America and I really want to get some sequences from this unique community.

I attempted to extract DNA by grinding the crust in a clean mortar with dry ice and then running though the MoBio soil extraction kit (which works great for our typical types of samples).

Nanodrop photospectroscopy indicates DNA at reasonable concentration (compared to other samples I've successfully worked with), but also seem to show absorbency at other wavelengths--perhaps indicating dissolved minerals that didn't get removed by the extraction kit?

So then the PCR issues:  with the 8F/1392 combo I'm getting light 1.4 kb bands with 40-43 cycles, but my negative controls also seem to be getting the light band.  On all my other projects this year (I'm pretty new at all this) I've had nothing show up in my neg control, and had great bands in my target reactions, furthermore cursory examination of my 2,500 sequence data base I just finished indicates only alpine soil bacteria (so no or very little contamination).  Of course, those reactions ran only 20-30 cycles...

Sorry to ramble but here now are my questions:  is there always a low level of ambient bacterial DNA contamination in labs that will show up with enough amplification, but usually gets swamped out when you use enough target DNA?  Aside from a PCR hood, UVing everything, cleaning with EtOh, should I use anything else?  Anyone use DNA Away for cleaning?

And then any recommendations on extracting/amplifying DNA from this rocky matrix, (or how to confirm there really is a permanent community of microbes there) would be much appreciated.

Thanks!

[phage434]

> 40 cycles will amplify something almost always.  Whatever is amplifying is about a million times less prevalent than your typical sample, which amplifies at 20 cycles.  A single cell might, in principle, provide this template.  In other words, this is almost certainly meaningless.

Your primers may not be amplifying the DNA from this sample.  More likely there is no sample, but I could be wrong.  You might try a bead-beater and glass beads to fractionate whatever there is that is there.

[303microbialist]

phage434, on Jun 2 2009, 05:02 PM, said:

> 40 cycles will amplify something almost always.  Whatever is amplifying is about a million times less prevalent than your typical sample, which amplifies at 20 cycles.  A single cell might, in principle, provide this template.  In other words, this is almost certainly meaningless.

Your primers may not be amplifying the DNA from this sample.  More likely there is no sample, but I could be wrong.  You might try a bead-beater and glass beads to fractionate whatever there is that is there.

Thanks a lot.  That's good to know, at least I won't be obsessively changing reagents and cleaning my whole lab in a futile attempt to get this to work.  I did use bead beating, which is part of the MoBio extraction kit... I think I'll have to go back attempt some assays on the parent material as well as the attempted extractions to search for DNA and or potential PCR inhibitors.  Not exactly sure where to start on that though...

[phage434]

Make sure you are not adding too much template to the PCR reaction.  Try diluting the sample 100x and 1000x, which will essentially eliminate inhibitors, but only delay the pcr by 7-10 cycles.

[AquaPlasmid]

303,

I'd be happy to send you some sample of our AquaStool reagent, which is very effective in removing PCR inhibitors in feces. The most thorough bacterial DNA extraction we have found is to combine beadbeating with sonication, which of course causes severe DNA shearing, but I think it wouldn't affect your cloning and sequencing, as your priority is getting all the DNA out and getting a fully represented library. Just polish the ends and then ligate.

Good lucks,

Aqua
www.aquaplasmid.com

[microgirl]

Which MoBio kit are you using? I also do environmental samples and have found the PowerSoil kit to be better than the other soil kit (whose name I can't remember). Another thing that I have found is that it is sometimes helpful to run your samples through Chromaspin columns after extraction - if you want to try this I can give you a generalized protocol. This is kind of a pain since you need to precipitate your DNA afterwards to concentrate it, but it does remove inhibitors left behind by the kit. I'm wimpy when it comes to ethanol precipitation - I always lose or are afraid of losing my pellet but PelletPaint helps with this.

303microbialist, on Jun 2 2009, 02:58 PM, said:

Hi all,
I'm hoping I can get a bit of advice on some issues I'm having with my current project:

I use "universal" bacterial 16s primers, 8F/1392 in this case, to amplify environmental soil DNA extracts for use to clone and sequence.

We found a seemingly biotic crust (bright green and yellow on a white hard base) at around 18,000 ft on a volcano in South America and I really want to get some sequences from this unique community.

I attempted to extract DNA by grinding the crust in a clean mortar with dry ice and then running though the MoBio soil extraction kit (which works great for our typical types of samples).

Nanodrop photospectroscopy indicates DNA at reasonable concentration (compared to other samples I've successfully worked with), but also seem to show absorbency at other wavelengths--perhaps indicating dissolved minerals that didn't get removed by the extraction kit?

So then the PCR issues:  with the 8F/1392 combo I'm getting light 1.4 kb bands with 40-43 cycles, but my negative controls also seem to be getting the light band.  On all my other projects this year (I'm pretty new at all this) I've had nothing show up in my neg control, and had great bands in my target reactions, furthermore cursory examination of my 2,500 sequence data base I just finished indicates only alpine soil bacteria (so no or very little contamination).  Of course, those reactions ran only 20-30 cycles...

Sorry to ramble but here now are my questions:  is there always a low level of ambient bacterial DNA contamination in labs that will show up with enough amplification, but usually gets swamped out when you use enough target DNA?  Aside from a PCR hood, UVing everything, cleaning with EtOh, should I use anything else?  Anyone use DNA Away for cleaning?

And then any recommendations on extracting/amplifying DNA from this rocky matrix, (or how to confirm there really is a permanent community of microbes there) would be much appreciated.

Thanks!

[303microbialist]

Yes, I use the PowerSoil kit...  What's the deal Chromaspin columns?  Thanks.

microgirl, on Jun 4 2009, 10:01 AM, said:

Which MoBio kit are you using? I also do environmental samples and have found the PowerSoil kit to be better than the other soil kit (whose name I can't remember). Another thing that I have found is that it is sometimes helpful to run your samples through Chromaspin columns after extraction - if you want to try this I can give you a generalized protocol. This is kind of a pain since you need to precipitate your DNA afterwards to concentrate it, but it does remove inhibitors left behind by the kit. I'm wimpy when it comes to ethanol precipitation - I always lose or are afraid of losing my pellet but PelletPaint helps with this.

303microbialist, on Jun 2 2009, 02:58 PM, said:

Hi all,
I'm hoping I can get a bit of advice on some issues I'm having with my current project:

I use "universal" bacterial 16s primers, 8F/1392 in this case, to amplify environmental soil DNA extracts for use to clone and sequence.

We found a seemingly biotic crust (bright green and yellow on a white hard base) at around 18,000 ft on a volcano in South America and I really want to get some sequences from this unique community.

I attempted to extract DNA by grinding the crust in a clean mortar with dry ice and then running though the MoBio soil extraction kit (which works great for our typical types of samples).

Nanodrop photospectroscopy indicates DNA at reasonable concentration (compared to other samples I've successfully worked with), but also seem to show absorbency at other wavelengths--perhaps indicating dissolved minerals that didn't get removed by the extraction kit?

So then the PCR issues:  with the 8F/1392 combo I'm getting light 1.4 kb bands with 40-43 cycles, but my negative controls also seem to be getting the light band.  On all my other projects this year (I'm pretty new at all this) I've had nothing show up in my neg control, and had great bands in my target reactions, furthermore cursory examination of my 2,500 sequence data base I just finished indicates only alpine soil bacteria (so no or very little contamination).  Of course, those reactions ran only 20-30 cycles...

Sorry to ramble but here now are my questions:  is there always a low level of ambient bacterial DNA contamination in labs that will show up with enough amplification, but usually gets swamped out when you use enough target DNA?  Aside from a PCR hood, UVing everything, cleaning with EtOh, should I use anything else?  Anyone use DNA Away for cleaning?

And then any recommendations on extracting/amplifying DNA from this rocky matrix, (or how to confirm there really is a permanent community of microbes there) would be much appreciated.

Thanks!

[303microbialist]

Ya, I'd love to try some AquaStool?  

AquaPlasmid, on Jun 3 2009, 02:55 PM, said:

303,

I'd be happy to send you some sample of our AquaStool reagent, which is very effective in removing PCR inhibitors in feces. The most thorough bacterial DNA extraction we have found is to combine beadbeating with sonication, which of course causes severe DNA shearing, but I think it wouldn't affect your cloning and sequencing, as your priority is getting all the DNA out and getting a fully represented library. Just polish the ends and then ligate.

Good lucks,

Aqua
www.aquaplasmid.com