Sequencing Problem...

[Mart Speek]

wetsel_r at wums.wustl.edu wrote:
: Greetings netters:

: This is probably a sequencing FAQ but here goes:  I have a technician in the
: lab that can do a "single" load reasonably well.  The problem is when the
: run is extended or even "double" loaded.  What we're getting is fuzzy
: , sometimes doubled bands which are difficult to decipher.  Is the Urea going
: off, temp too low/high, any suggestions would be appreciated...

: OH! BTW - I should note that this is double-stranded sequencing with oligos
: and using Sequenase.

: Thanks in advance,

: David
: haviland at kids.wustl.edu


Dear David;

I have noticed the same phenomenon when using commercial SEQ 
apparatus (Macrophor, LKB), but never with home-made machines and
gels run with contact with metal plate. So, I propose this is
some kind of combination of heat and high voltage (thermostbilized
plates) that makes longer runs difficult or impossible to read.
Indeed, when reducing voltage (down to 1 kv) bands appear to be
sharper. I hope this explanation helps you somehow.

Cheers,

Mart (mspeek at ebc.ee)(ESTONIA)