Dino

Hi everyone,

Just wondering for those who have experience with sequencing. Does having RNAseI in your plasmid preps affect sequencing?

I have a bit of contam in my samples and a little paranoid it will not give me clean sequencing.

Thanks!
D

Hi All,

I've been trying to clone a gene into the pGEMTeasy vector and was successful in getting colonies and isolating plasmid.
When I perform PCR using my target gene primers I also get a nice clean band.

When I initially plasmid prep'd the samples using a low copy vector protocol (to get a higher yield)
the negative control (a background colony, blue) always ends up with a significantly higher concentration
around 150-200ng/uL however the clone always purifies at 60-80ng/uL.

I've tried this on two different occassions, each time purifying two clones and one background colony.

Any ideas as to why I'm getting low yields for the clone and not for the background colonies?
I'm using DH5alpha home made and the vector is commercial.

Could the insert going in backwards have any effect on plasmid copy number?

Thanks!
D