Transformants pictures in gel - (Aug/04/2008 )

Hi
I did transformation So I have tried to cloned my insert into a pJB785TTKm1 vecotr(Promoterless vector for protein expression). its size is 7.6 Kb and my insert is 650 bps. To screen, I made plasmid preps from mu putative clones and as you know plasmid is supercoiled. I run the plasmid preps from those colonies and I see multiple bands as my vector but one of those bands are a little bit higher. I don't know how I can find that is my real clone or not??
I Know as the next step I can do restriction digestion or PCR on my plasmid preps.
Please let me know your ideas
Thanks
bernard

Are you saying that you are running uncut plasmids out on a gel to determine which is the correct clone?? This won't work, you can't determine your plasmid size from an uncut plasmid. You need to do a restriction enzyme digest to linearise your plasmid if you want to determine size.

Hi Bernard,

My suggestion would be to screen initailly by PCR and then confirm by restriction digestion and later sequence confirm if necessary.

Screening your plasmids by PCR would be a foolproof method. Use a combination of vector specific primer and insert specific primer so that you can screen for orientation of the insert also.
Eg - vector Forward and Insert reverse

Then you can confirm by digestion using the enzymes you used for restriction cloning.

Regards
Avinash

http://aviprem.gq.nu