pulse-field gel ......

POPOVA ANDREI.POPOV at afrc.ac.uk
Sat Dec 3 13:34:43 EST 1994


>I am having problems in resolving DNA fragments when running the PFG.
>These problems do not appear to be due to the quality of the chromosomal
>preps since yeast markers and low molecular weight markers purchased as
>standards also resolve poorly.  The PFG's are run on a Beckman TAFE
>Geneline system.  All of the runs have been at a constant
>current-initially at 170mA for 30 minutes with switch times of 4s and then
>150mA for 18 hours at a switch time specific for the size of the DNA
>fragments I am looking for. I have been using Beckman LE pulse-field grade
>agarose at a concentration of 1%, and running in 1X TAFE buffer (which is
>approximately the same as .4X TAE).  The temperature at which the gels are
>run is app. 12-14 C.  I have tried to correct the problem by trying a
>number of different things. 


HI STEVE,
I WOULD SUGGEST TO USE:
1. 0.5 x TBE (IN MY HANDS RESOLUTION WAS BETTER THEN WITH 0.5 x TAE)
2. HIGH STRENGTH AGAROSE (I USE BOER. MANN. MP AGAROSE)
3. LOWER THE TEMP. TO 3.5C
4. FOR 3000 ML OF 0.5 x TBE WE USE 150-200 V DEPENDING ON THE SWITCH TIME
  ; THE HIGHER THE SWITCH TIME THE LOWER SHOULD BE YOUR VOLTAGE (AND POWER)
   

18 HRS RUNNING TIME SUGGESTS THAT YOUR PFGE TAKES APPROXIMATELY HALF OF 
 THE TIME THAT IS NEEDED TO RESOLVE 1 MB FRAGMENT....

ANDREI




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