Biology Reference
In-Depth Information
Pristionchus pacificus (George Hsu, Heather Roberson, G.B.-M. and M.M.,
unpublished observations) with species-specific probes. One consideration for
embryonic staging is the degree to which embryos are retained by adults. In our
hands, C. remanei (strain PB4641) and C. briggsae (AF16) retain fewer embryos
than C. elegans. Possible solutions are to limit the amount of food to promote egg
retention; use of a mutant background (e.g., Egl) that retains eggs (we have used
ir12, an uncharacterized recessive dpy mutant of C. briggsae with good results); to
bleach gravid adults and let the isolated embryos develop for several hours; or to
bleach to isolate embryos from a plate. In our experience, bleaching solution
negatively affects staining quality, so it is recommended to use a diluted bleach
solution and rinse animals thoroughly. For consistent freeze-cracking, adults can
be added back as suggested above.
VIII. Troubleshooting Guide
Problem
Cause
Remedy
Lack of signal
Lack of template for
transcription
Confirm amplification of PCR product
with gel electrophoresis.
Error in T7
sequence
Confirm that primer sequence was correct
as ordered; subclone PCR product into
T/A vector and sequence it; be certain
correct (reverse) primer had the T7
sequence, not the sense primer
Wrong synthesis kit
or components
Confirm use of all components from a
DIG-UTP kit, not DIG-dUTP (used for
DNA probes)
Poor transcription Run side-by-side aliquots of (+)T7 and
(
)T7 transcription reactions to
confirm synthesis of RNA
Developer problem Spot the probe onto a blotting membrane
(e.g., nitrocellulose or Hybond-N+)
and test for color turnover. Try a
previously successful probe. Remake
developer components and solutions.
(It is recommended to include a
positive control in all experiments to
rule out problems with the reagents or
solutions.)
(Continued)
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