fluorescent detection: microsatellites (help)

dbell dbell at qnis.net
Thu Sep 6 11:39:50 EST 2001


Hi Eric

Unfortunately there are no "easy solutions"

I have done some small amount of microsatellites on my ABI 310.
My general recipe for PCR: 1X buffer, 1 unit PE amplitaq gold (no affil, 
but IMHO, i can always count on it), 1.5 - 2.0mM MgCl2; 200uM ea dNTP; 
20pmoles ea primer; 10 - 100ng DNA.

You will need to do a MgCl2 gradient to optimize PCR

Annealing temperatures for your primers should also be optimized

You can also play with the number of PCR cycles

What is the fluorescent dye on your primer?  I have found the 310 to be 
very sensitive to the blue and green dyes, but I do not get good results 
with the yellow dyes - my standard is red. You can also vary the amount of 
PCR product you add to the diformamide, I have tried anywhere from 0.5ul to 
2.0ul.  This is a relatively easy gradient to try.

Sometimes you just get a run that is bad :-(

Hope this helps - feel free to contact me for more help
Deanne Bell
USDA Agricultural Research Service
2021 South Peach Avenue
Fresno, CA 93727
(559) 453-3170
(559) 453-3088 fax

On Tuesday, September 04, 2001 7:34 AM, ParentE at dfo-mpo.gc.ca 
[SMTP:ParentE at dfo-mpo.gc.ca] wrote:
: Hello all.
:
: We have a problem with fluorescent primers and would need advice. We have
: been doing microsatellites work on snow crab for awhile now and never had
: any (not much) problem using radioactively labelled primers.  We recently
: purchase an ABI 310 automatic sequencer and are in the process of 
quitting
: radioactive work for fluorescent primers.  The problem starts here. For 
some
: loci we didn't had any problem, but in some case we can't get any signal.
: The weird thing is that we tried to labelled one primer with 
radioisotopes
: and use the fluorescent primer as reverse. We get a good signal on
: acrylamide. When using the same primers set but without radio labelling 
we
: don't get any signal on the sequencer. Should we try to re optimise the 
PCR
: or is there a simple explanation to our problem?
:
: I must admit that I'm baffled by all that and need advice very quickly
:
:
: Thank you in advance for your help
:
:
: Eric Parent
:
: Maurice Lamontagne Institute
: Fisheries and Oceans Canada
: 850 route de la mer
: C.P. 1000
: Mont-Joli, Qc
: Canada
: G5H 3Z4
:
: parente at dfo-mpo.gc.ca
: http://www.qc.dfo-mpo.gc.ca/iml/
:
: ---
: 

---




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