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]
|