Dna cloning using NdeI/BamHI using pET11 vector - (Oct/14/2010 )

Hi all,
Thia is my first post. Can someone help me with ligation?
I have cut my vector (pET11) using BamHI/NdeI and I have got single band. I think its working.
I have cut my insert with the same enzymes. I got three bands on gel. This was because of internal BamHI site in my insert. So to get rid of it I am going to do quick change mutation. But meanwhile I took the expected (300bp) insert and tried to do ligation keeping ration between vector to insert as 1:6 (molar). Almost tried 5 times with different combination but no clones. Can someone help me. I have get this work done soon.
Sowmya

and why do you try 1:6 and not 1:3 (molar)?
just a suggestion!
good luck!

check if your insert is able to ligate with itselfe ...do a ligation reaction with just insert and ligase (+buffer) and put it on an agarose gel ...you should see somethink like a ladder if everthing is okay. NdeI can be a really nasty enzyme ...make sure that there are enough nucleotides upstream of your recognition site. For details see this link.

Regards,
p

rota on Fri Oct 15 14:46:49 2010 said:

Hi,
Thanks for the suggestion. Today I am going to try with 1:3 ratio

pDNA on Fri Oct 15 16:56:55 2010 said:

Hi,
As I have used different enzymes, self ligation may not be a problem. Recognition site is not a problem. I do get the intended insert size after digestion. I was just thinking about something to do with ligation. I suspect that I have exposed the DNA too much to UV.
Just had another question. Is there any use in doing TA cloning and then excise the region of interest using the restriction enzymes. I am working for a amelogenin mutant. Let me quote the reference which I am using J Dent Res 78(3): 743-750, March 1999. Thanks a lot for your suggestion.