Non-radioactive blots -- Responses
sjclough at uiuc.edu
Wed Sep 18 16:54:44 EST 2002
A few weeks back, I asked the group about the use of non-radioactive
kits for Southerns and Northerns. I received 7 responses. 5 were in
support of Roche's DIG-labeling kit. I also received a vote for
Pierce's North2South Kit and a vote for Amersham's ECL kit.
Summary of suggestions/comments on Roche's DIG system included:
* For Southerns, use the PCR-based kit.
* For Northern, use the Sp6/T7 RNA transcription kit. If probe is
from a plasmid insert, be sure to cut insert free of plasmid and gel
purify. If one attempts to transcribe off a plasmid, could have the
entire plasmid transcribed and labelled.
* Their random-priming kit does NOT seem to work as well as the PCR
or transcription kits
* PCR labelling works best if probe is less than 1 kb. For larger
probes, it works better if the DIG-dUTP label is diluted. PCR seems
to be somewhat inhibited by the DIG-dUTP.
* Works best if use all Roche reagents and membranes.
* Be sure that the membrane is VERY wet when adding the
chemiluminescent substrate or you'll get
* Compared to P32 , the amount of target on the membrane might need
to be increased (one wrote: we use about 3 ug of fungal DNA for one
lane on a southern, which I think is kind of high for a genome size
of 30 Mb)
*Their manual is very helpful and is available on-line
I guess I should add the disclaimer that I have no connections to
Roche, Pierce, or Amersham. I'm simply passing on comments from
those that responded to my request on non-readioactive labelling kits.
Steven J. Clough
Research Geneticist, USDA-ARS
Assistant Professor, U of Illinois, Crop Sciences
1101 W. Peabody Dr., 236 NSRC
Urbana, IL 61801
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