Mortality during P-element transformation.

[Slawek]

Dear Netters,
I've been doing injections of Drosophila embryos with P-elemnt based 
constructs.  After gaining some practice I used to have an efficiency of 
the hatching about 30 - 50%.  I haven't been using the technique for a 
couple of months.  When I started to inject embryoes again the hatching 
rate dropped up to 0%.  I started to test all possible reasons of my 
problems.  I found following things:
1. When I test undechorionated eggs I get 15-18 larvae out of 20 eggs.
2. I get the same rate of hatching when the same eggs are covered with a
   layer of oil up to 4mm.
3. When the eggs are dechorionated (I dechorionate them with sticky tape)
   and covered with oil only 0-5 larvae appear.  Sometimes unhatched eggs
   seem not to be developed at all, on other days they are quite
   advanced in the development.
4. I use the same equipment, sticky tapes and oils.  I checked influence 
   of following factors:
   a) chemicals used for preparing agar plates on which I keep embryos after
      injections (agar, sucrose, water) were used from different bottles;
   b) 2 stockes were tried: w,+,TM6/Sb e delta2-3 and w,+,delta2-3/delta2-3;
      they give the same results, previously I used the first one;
   c) the width of the sticky tape on which eggs are positioned doesn't have
      any influence;
   d) the light of the microscope under which I dechorionate embryoes seemed
      to be very strong, but turning it off and tensing my eyes didn't help.
   e) two different kinds of oil, either saturated with water or not give 
      simmiliar results.
5. Humidity in a room in which I used to work is now much higher then before,
   but it could eventually make things more easy.  When I test an influence 
   of any  factors I simply dechorionate eggs and immediatelly cover them 
   with oil.
Could anyone have any suggestions about these problems?  Please send letters
to the newsgroup or immediatelly E-mail to me.

Slawek Bartoszewski
E-mail: bartoszewski at rsbs1.anu.edu.au