No apology necessary Susannah. Everybody has its own preferences when it
comes to Molecular Biology. If "no BSA" works fine for your
applications, then everything is fine, and you should keep it that way.
I think that the people who need it the most are people who need to
fight against high amounts of Humic Acids. BSA is great because it is
much cheaper than other reagents like the T4.
I agree though that people should try to mention it more often in their
publications. We are so used to our good old protocols that we tend to
skip more and more the M&M part. Or maybe it's the journal's fault.
Nowadays, the shorter, the better, sometimes it's a pity. Especially for
people who recently started working with degraded DNA.
Now, I'm on a roll people. I have a last question for the Centricon
lovers.
As you probably know, Millipore stopped making/selling centricons.
What are you using instead? Ultra-4? Vivacons? What is your experience.
For those who work with human DNA, did you detect contamination
problems?
I really like the ultra-4 but I start thinking they might be slightly
contaminated with very small human DNA fragments.
-----Original Message-----
From: Ancient DNA List [mailto:[log in to unmask]] On Behalf Of
Susannah Baldry
Sent: Wednesday, October 29, 2008 5:41 PM
To: [log in to unmask]
Subject: Re: BSA
I apologise, I didn't mean to make it sound like a UK "consensus"! Just
a consensus of the people still in the office at 5pm! I should clarify
that our team works primarily with modern DNA and we do not have any
trouble with our reactions omitting BSA. I believe the high-throughput
sequencing teams do use BSA as part of the commercial kit; I am not sure
why it is required in their chemistries but not our small-scale
experiments. I suppose modern samples are more simply cleaned than aDNA
so inhibitive compounds are not such an issue.
However I stick by my original statement, that if a paper details the
contents of a PCR reaction and does not mention BSA, then BSA should not
need to be assumed!
Susannah
Frederika Kaestle <[log in to unmask]> wrote:
> Actually, we still use BSA a lot in the US in our ancient DNA work.
In
> the past (when I was in grad school in David Glenn Smith's lab), we
did
> some in-house experiments and found it does help with problematic
> extracts that seem to inhibit enzyme (Taq) activity due to coextracted
> compounds. Since it's no too expensive, we just add it to all our
aDNA
> reactions (unless we're working with ancient bovids, in which case we
> use Rabbit Serum Albumin). We haven't had a problem with it becoming
> contaminated, although in my lab our stock is aliquoted into very
small
> volumes. But if the consensus in the UK is that it's a waste of time
> and money, I'd love to see some research showing that.
>
> best,
>
> Rika
>
> Dr. Frederika Kaestle
> Departments of Anthropology and Biology
> Indiana University, Bloomington
> USA
>
> Susannah Baldry wrote:
> > Dear Odile,
> >
> > I am not sure which protocols in the past used BSA in the PCR mix,
but
> > (after throwing your question at my office colleagues) we agree it
is
> > not common now - except for some commercial enzyme kits for arrays
and
> > sequencing. In the latter case you would be following manufacturer's
> > protocols which would be exhaustive. If a published paper specifies
> > the contents of the PCR reaction it should be complete & repeatable,
> > so you will be safe following their protocol as described.
> >
> > Good luck with your experiments,
> >
> > Kind regards,
> >
> > Susannah Baldry
> >
> >
> > Odile Loreille wrote:
> >> Hi everyone,
> >>
> >> I'm back with a second question. Reading the latest aDNA papers, I
> >> noticed that many PCR protocols don't mention BSA anymore.
> >> I was wondering if you, aDNA specialists, omitted this information
> >> because you believed that the presence and amount of BSA isn't
worth
> >> mentioning or if you truly stopped using it in your
amplifications.
> >>
> >> If you want your response to be private, please simply write to
> >> [log in to unmask]
> >>
> >> Thank you very much
> >>
> >> Odile
> >>
> >
> >
> >
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