At 07:30 AM 10/27/00 -0600, you wrote:
>Thank you very much, I am going to try this.
>Rhonda
Dear Rhonda,
also note Sheryl Christoffersons message; Ppt. of borates actually reduces
peak resolution mid sequence rather than causing a decline in absolute
signal strength so with hind site it may be that Sheryl is closer to the
mark; Incidenatally, I too have seen such a decline following a sring of
T's : incorporation of T's is a recognised problem with big dyes which is
why big dyes actually utilise uracil and not thymidine. No such problem
however pertains to dRhodamine which therefore does incorporate thymidine
and not uracil.
Accordingly, if my suggestion doesn't work try the folowing :
1. double the amount of big dye and up the number of cycles in your
sequencing reaction by 5 say from 25 to 30 : I have personally seen this
cure dropout t's.
2. Alternatively, try the increased number of cycles with d-Rhodamine but
use twice the volume of this chemistry as compared with big dye ( in
essence, big dye is a more sensitive version of dRhodamine by virtue of
being a dRhodamine-fluoroscein conjugate ).
3. As a final resort play with the extension and/or annealing temperatures :
Try extending at 55 for instance instead of 60 and adding an extra 1mM
Mgcl2 to the sequencing mix. The latter increases the processivity of taq
and therefore its ability to navigate difficult regions.
Hope this helps and let me know how you get on.
Best wishes,
Laurence S Hall
>-----Original Message-----
>From: Laurence Hall [mailto:lhall@megabase.aecom.yu.edu]
>Sent: Thursday, October 26, 2000 1:52 PM
>To: Polchak, Rhonda
>Cc: Recipients of ABRF List
>Subject: Re: DNA troubleshooting
>
>
>At 12:12 PM 10/25/00 -0600, you wrote:
>>Dear ABRF Members,
>>I run an ABI 377 DNA Sequencer and use the software from ABI. A sample
>>submitted to our facility sequenced well to ~400 bases and then the signal
>>was completely lost for ~50 bases. ( A series of N's in the
>>electropherogram). The signal was then restored for the remainder of the
>>sequence. Does anyone know why this would occur?
>>Thanks in advance.
>>
>
>
>>Rhonda Polchak
>>Sequencing Core
>>Heska Corporation
>>Fort Collins, CO 80525
>>polchar2@heska.com
>>
>>Dear Rhonda,
>
>loss of resolution is always a gel/plate problem and this particular
>problem is often attributable to ppt of borates in TBE; My advice :
>
>1. Try switching your plates for running if this is an ongoing problem or
>certainely soak your existing plates in 1M KOH/methanol for 30minutes
>followed by copious washing in water.
>
>2. Infact, you may want to routuinely add a boiling water flush to your
>cleaning plate procedure.
>
>3. Definitely, make up fresh TBE !!
>
>Hope this helps !!
>
>
>Laurence Hall
>>
>****************************************************************************
>**
>
>Laurence Hall,
>Einstein Genome Centre,
>1695 Poplar Street,
>New York 10461.
>
>Tel. (00) 1 718 405 8380
>
>Fax. (00) 1 718 405 8383
>
>E-Mail : LHall@megabase.aecom.yu.edu
>
>
>****************************************************************************
>**
>
>
******************************************************************************
Laurence Hall,
Einstein Genome Centre,
1695 Poplar Street,
New York 10461.
Tel. (00) 1 718 405 8380
Fax. (00) 1 718 405 8383
E-Mail : LHall@megabase.aecom.yu.edu
****************************************************************************
**
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