Yes, I already ordered the primers. I hope it will work.
Thanks Eske.

-----Original Message-----
From: Ancient DNA List [mailto:[log in to unmask]] On Behalf Of
Eske Willerslev
Sent: Saturday, October 18, 2008 10:34 AM
To: [log in to unmask]
Subject: Re: Direct sequencing of very small amplicons

You should have a look at :

Binladen J, Gilbert MT, Campos PF, Willerslev E.

5'-tailed sequencing primers improve sequencing quality of PCR products.
Biotechniques. 2007 Feb;42(2):174, 176.

Eske


On 10/16/08 4:17 PM, "Loreille, Odile CONTR"
<[log in to unmask]>
wrote:

> Hi Sarah,
> 
> You're right, this is frustrating. I think I will have to wait for the
> pyrosequencer to arrive. We should have received it months ago, then
the
> order was cancelled. So I'm not sure whether we'll ever get it.
> 
> The problems I had with my last short amplicon (which was 85bp total)
> was the short poly C stretch (see jpeg).
> I'll try a dilution series of the PCR product and play with the
> injection time a little until I get longer primers and a
pyrosequencer.
> We'll see.
> 
> Thanks a lot for responding to me, I really appreciate.
> Perseverance is my best asset -:)
> 
> All the best,
> 
> Odile
> 
> -----Original Message-----
> From: Ancient DNA List [mailto:[log in to unmask]] On Behalf
Of
> Palmer, Sarah
> Sent: Thursday, October 16, 2008 6:02 AM
> To: [log in to unmask]
> Subject: Re: Direct sequencing of very small amplicons
> 
> Hi Odile,
> 
> I too am in a very similar predicament. I have had some success with
> direct sequencing <70b amplicons, although quite limited... I have
been
> aiming for 2ng of PCR product in a 20uL big dye reaction. The most
> successful sequencing I have achieved was by making an 80uL mix with 8
> ng PCR product and dividing the mix across 4 tubes. However, even with
> this I generally got 1 or 2 in each 4 that gave me something
meaningful
> and then I had to read in the sequences from the chromatograms. Some
of
> the problem at the output end is that the sequencing software is
simply
> not designed to call short sequences. It gains a 'confidence' in
calling
> the signals based on the number of them in the sequence, so don't be
> afraid to edit. The next strategy we are going to try is cloning these
> short amplicons in order to have something longer to sequence.
> 
> I hope some of this helps. Perseverance is the key. It's comforting to
> know I'm not the only one out there. Let me know how you get on.
> 
> Good luck,
> Sarah Palmer
> 
> 
> -----Original Message-----
> From: Ancient DNA List on behalf of Odile Loreille
> Sent: Wed 10/15/2008 6:59 PM
> To: [log in to unmask]
> Subject: Direct sequencing of very small amplicons
>  
> Hi Everyone,
> 
> I need to "direct sequence" a 59bp amplicon and so far my data look
> awful.
> I use the ABI big dye (1.5ul for a 20ul reaction), 0.5ul of dGTP,
20pmol
> of 
> primer and still 25 PCR cycles. I still need to test different
dilutions
> of the PCR 
> product.
> 
> Does anyone have any experience with modified conditions that would
help
> 
> decrease the background?
> 
> Any advice would be appreciated.
> 
> Cheers
> 
> Odile
> 
> 
> Dr Odile Loreille
> Senior Research Scientist
> Armed Forces DNA Identification Laboratory
> 1413 Research blvd
> Rockville, MD 20850
> USA
> [log in to unmask]
> 

-- 

Prof Eske Willerslev
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