Cloning a 2kb fragment! - (Jan/03/2006 )
I have been unsuccesfully trying to apmlify a 2kb fragment by PCR and it's just not working! The thing is that the plasmid with my fragment was prepared by a student whose DNA work was really bad. Trying from the over night culture doesn't help because I am not getting any fragment around 2kb at all. I've tried washing pellet with PBS and stuff but still nothing. Even when I get something with PCR, it gets lost by a round of ethanol precipitation.
I've tried DMSO and BSA and both gave no better results then without adding them at all. What is your experience with other adjuvants, like formamide, betaine and even glycerol?
If possible, re-do the original cloning... or re-transform e. coli with the plasmid from the student.
If you still have some PCR product, you can dilute this very much and try a second round of PCR with the same primers?
vairus is right. You will lose a lot of time and patience trying to get something from bad samples.
try to go back far enough to skip all the steps that the student did.