-dilution of "Big Dyes"

Woodland, M P m.p.woodland at ic.ac.uk
Wed Dec 20 04:35:22 EST 2000


Hi -  I  use  a  377  in  a  small  core  facility  and  routinely  dilute
the  BIGDYE  v2.0  by  2x  and  use  a  10ul
reaction  volume  and  purify  the  sequencing  reactions  with
Amersham/Pharmacia  spin  columns.
 To  gain  maximum  reads  I  use  the  ABI  Long  Read  protocol. - I
could'nt afford  to use  this  set  of 
protocols  unless  I  diluted  the  BIGDYE ! For  the  m13  control  just
this  Friday  I  achieved  just  under IKbp
  with  4Ns - this  is  not  unusual - not  always  the  case  foe  samples.
If  a  sample  sequences  poorly  I  use 
 the  std  protocol- although  this  rarely  appears  to make  a  great
difference. One  aspect  of  the  v2.0  is  that  the  front  end  is  much
cleaner. I  only  use  DMSO
with  GC  rich  as  I  have  always  understood  that  TAQ  pol  does'nt
like  it. When  the CORE  kits  were  available
I  always  use  to  add  more  TAQ  halfway  thru  the  GC  rich  cycles  to
extend  the  read  length.

What  is  different  about  capillary  based  systems  that  dillution  and
v2.0  apparently  gives 
 such  poor  results?

Regards

Marc

M.P.Woodland Ph.D
Imperial College
Uni. of London
U.K.


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