I am a student in K-JIST in Korea.
We've got several embryonic lethal mutations near the Unc-29 region on
LG I.
[ unc-29(e193) Let/ dpy-5(e61) unc-13(e1091) ] I.
Currently the possible mutation region was narrowed down around 15
cosmids.
Now, I'd like to find out a cosmid containing lethal mutation by
germ-line transformation.
I am familliar with microinjection technique however, I am really
worrying ahout this rescue experiment because of my short
knowledge and experiance. Therefore I would appreciate if you could
give your great information to me.
If you are willing to help young student, please answer fallowing
questions.
1. There are several overlapping cosmids containing same sequence. Is
there any criteria in selecting cosmids?
2. Could I get a protocol for cosmid preparation? Do cosmids need
special purification procedure for injection?
3. What quantity ratio would be suitable between cosmids and marker
plasmid(rol-6) in injection mixture?
4. What kinds of control experiment are possible? (i.e. rol-6 only
injection)
5. On which generation should I observe the phenotypic rescue after
cosmid injection?
6. In our mutant strains, recombination is so frequent that the lethal
mutation is often disappear.
Therefore, how can I distinguish rescue of lethality from this
recombination after cosmid injection?
7. Could you recommend some references which can rescue me from
obscurities?
