hi all, i am trying to clone a PCR amplicon into a vector. in both amplicon and vector, i first need to endfill it (in the amplicon to get a blunt end and in vector i have to first cleave with a sticky end cutter and then end fill it to inactivate the site, as i still have more ligation steps) and then ligate them. i am trying it since some time, but i aint getting the desired clones. i know, its a process controlled by nothing so to say.

i need to explain why i am not getting the clone in first shot or in intial ligation reactions, can anyone please help me to get refrences from books or journals to explain this rate limiting stem in molecular biology.

please reply fast, i need to present a project status report shortly.

PLEASE HELP.

Hi ecolik12,

Sorry but without more information it is almost impossible to say why it hasn't worked first go. Have you included controls to help deduce where the problem may be? Blunt end ligations can be fiddly at the best of times, do you check DNA concentrations on a gel before ligating and then use appropriate insert/vector ratios I tend to try and really drive blunt end ligations with a ratio of around 10/1 (gives a few concatomers but usually can pick a single insert.

So I guess the main points pertinent to you is:
1/ Efficiency of blunt end
2/ Efficiency of DNA recovery from Klenow treatment
3/ Concentration of DNA in ligation
4/ Efficiency of transformation

Cover these points with appropriate controls and you should be fine.

Sorry I can't help more.

Scott

hi,
one thing which i don't understand is that why haven't you used Pfu or Pfu turbo polymerase, Pfx,Pwo etc. which produce blunt ended product it self.
these things will decrease your DNA loss.
further you can also use PEG to increase efficiency of your blunt end cloning.
best wishes
amit