You write:
=> We are getting a stretch upgrade to our 373 in a couple of weeks and I
=> would be grateful for any tips users have.
=>
=> I currently run 85% M13 clones (PEG/phenol preps) and 15% pcr products
=> (PEG/NaOAc preps) all with ABI Taq FS terminators. If possible I do not
=> want to take on a new prepping method to get long reads on the 48cm wtr
=> plates as the current ones are high quality, quick, easy, reliable and
=> cheap. Will this quality of prep be OK for long reads? Do I need to
=> change the sequencing reaction conditions to favour longer products?
=>
=> Also I know the gel will have to be 4.75% (4.25%?). I currently use
=> sequagel-6 which is great. Do you recommend any particular ready mix
=> for the long gels?
=>
=> Finally can anyone give me some typical read lengths for the stretch
=> machine.
=>
=> Thanks in advance
=>
=> Kevin
=>
=> Kevin Clark |
=> kclark at immsvr.jr2.ox.ac.uk | If you havn't seen God, you're not
=> Institute of Molecular Medicine| driving fast enough.
=> Oxford,UK. |
=>
Kevin,
You might want to try the LongRanger Gel Mixes from FMC as they
seem to be more stable that the usual mixes and thus the matrix doesn't
degrade with the longer runs.
Also, as usual, you should do a DNA concentration curve to determine
the amount of template necessary for optimal, long reads.
Personally, until ABI comes out with better base calling software,
you'll have to manually correct the sequence out past 450-500 bases as
their software still hasn't got the base spacing correct and you'll see
great signal (if you have optimal reaction conditions with the Taq-FS)
but the calls will not be correct far out. You also might first want
to just use the normal length plates and optimize the conditions for that
(because you're used to pouring those plates) and then later give the
long plates a try. We did that but because the base calling was not so
good far out, continue to use the normal plates in the stretch upgrades
of the 373's. By the way, IMHO, the main advantage of the stretch upgrade
is that you get the more focussed lens and the longer well to read length
with the same plates.
Finally, our latest protocols are all available on my Web site:
http://dna1.chem.uoknor.edu
and it includes how we make our gels, alternative sources for plates, etc.
Cheers........bruce
***************************************************************************
Bruce A. Roe, Ph.D Department of Chemistry and Biochemistry
University of Oklahoma, Norman, OK 73019-0370, U.S.A.
Phone: (405) 325-4912 or 7610; FAX: (405) 325-7762; e-mail: broe at uoknor.edu
********************** http://dna1.chem.uoknor.edu/ ***********************
