I am a researcher in the Dept. of Life Science (Developmental Genetics
Lab.) in Kwangju Institute of Science & Technology, Kwangju, South
Korea. I would like to ask you certain questions regarding the problems
we faced during screening the C. elegans deletion library (EMS
mutagenized) by nested PCR.
This was our first attempt in mutant screening for ten genes (following
the protocol of R. Barstead) and we succeeded in identifying 5 positive
signals after the first round. All conditions were carried out as per
the protocol, where we used 75mM for carrying out the mutagenesis.
However, we lost all signals by the end of the second of sib selection,
while 2 others lost after the second sound sib selection with very weak
deletion band. Could you suggest the cause for the deleted bands to get
weakened in the successive screen? Additionally, in one particular case,
we observed a good number of males emerging in some plates after
mutagenesis. Can this be one of the reason also for losing the mutant
line as a several crosses?
We are preparing for a second attempt in the EMS mutagenesis, and before
doing so, we would really appreciate if you kindly give some valuable
suggestions and your opinions about our procedure and results. Your
experience and suggestions will surely help us to modify our technical
problem (if any). We hope to succeed this time.
Thank you very much for your patience.
Looking forward to hearing from you soon.
Name in Full: Shin, Ji-Yeon
Date of birth: May 6. 1973
E-mail: lifeinkj at geguri.kjist.ac.kr
Department of Life-Science
Kwangju Institute of Science and Technology
1 oryong-dong, puk-gu