SSCP-reproducibility problem

[Alkendi]

hi DrDNA

I am currently working on the 100bp DNA ladder (dsDNA) and it should produce 12 peaks on the CE. yesterday the profile showed 12 peaks!!!but all students in the lab are facing the problem of reproducibility, the result is not reproducible. After i got the sucssessful result I ran the same sample once again under similar conditions and I only generated one peak!! and I tried again after i changed the buffer and still one peak shows up.

By the way I am running the ladder in buffer only without polymer.

what could be wrong, I cannot get reproducible result for the same sample under similar condition??!!! which is very strange.

if that would help you think with me I use a flourescen dye (OliGreen) to tag the DNA and it is used for both dsDNA and ssDNA. In this case i am using it to tag a dsDNA. the process is I add enough amount of the dye to the DNA (1:1 ratio) and leave it for 5 minutes then run it on CE. During the 5 minute wait I wrap the tube with aluminum foile as the dye is light sensitive.

 

please think with me, what could be that I did not fix???

 

appreciate your help