I am doing drosophila embryo transformations using a cDNA clone. I am
getting poor or no survival of Ist instars when I did not even injected
with injection buffer. The exact method which I follow is....I discard
Ist 1 hr collection of embryos and collect next batch of embryos for
20-30 min. I dechorinate them in 50% chlorax (treatment for 70-80 sec)
and wash in deionised water for at least 8-10 time, just to make sure that
there is no trace of chlorax on the embryos. Then I give a gentle wash
with sterile DD water. I place the healthy embryos on double stick tape
using a fine brush. Double stick tape is from "Scotch" Cat.136. After
placing the embryos on tape I dessicate for 11 min (which is appropriate
for the existing humidity conditions). After dessication I place
halocarbon oil (series 700) on the embryos and am leaving the slide in
moist plastic box at RT (22 C). Even after 48-60 h I do not see at least
10% hatching. When I looked them under microscope time to time during
embryogenesis, they were perfectly completing the development in about 48
h and you can see the developed larvae moving in the vitelline membrane.
After this step none of them are emerging and they are dying.I am also
checking the oil on the embryos from time to time. One thing I noticed
about tape in the oil is, it is turning in to thick milky white color
after 48 h (i.e., almost at the time of completion of embryogenesis). I do
not know where the problem is lying. Is it the brand of doulbe stick
tape I am using responsible or some thing else? As I mentioned these embryos
were not even injected.
I greatly appreciate suggestions and comments from netters.
If any body has recently used particular brand of double stick tape which
worked well for them, I appreciate if they can send me a small portion of
the tape as a gift.
Thanks in advance
Dept of Biology
Colorado State University
Fort Collins, CO 80521