simple questions to answer - questions (Nov/19/2010 )
I have an exam next week and iam confused about some issues, Could anyone try to answer simple questions for me please:
We did an experiments in our lab. during the last 6 weeks about isolating RNA then generate TOP fragment by RT-PCR. After that we cloned the fragment into a plasmid ( PJET1.2) then we introduce the plasmid to a host. last step was to isolate the plasmid and digest it with restriction enzymes.
1- During the ligation prosses, what is the different between negative ligation and positive ligation?
2- Why we use negative ligation and if we have colonies on the plate, what is that mean?
I've never heard the term "negative ligation". Could you mean you did two ligations, one with your fragment included in the ligation mix (positive) and one without your fragment (negative)?
Yes yes i think what did you say is correct. the question will be ( if the plasmid carries ampicillin resistance and we use plate with ampicillin, why i should not have colonies there (( as our dr. said)).
Whether or not you can expect colonies to arise on ampicillin plates after transforming the host with your negative (no fragment) ligation control depends on how you prepared your plasmid before ligating. You said you "cloned the fragment into a plasmid". How exactly did you do this? What enzyme(s) did you use?