Ligation failure - (Jun/17/2008 )
HI
I have done reverse PCR of a 6 kb plasmid using mix polymerase. I used the 5' phosphorylated primers for blunt end ligation. BUt after ligation i am not gettion any colonies. My ligase, plates, competent cells are good and working for other cloning work. But this is not working . can anybody expalin the cause of failure. If any one has suffered such problem in the past and overcome it by some methods. please share your ideas with me.
sunil[color="#000080"][/color][size="5"][/size]
-sktewary-
QUOTE (sktewary @ Jun 18 2008, 03:55 AM)
HI
I have done reverse PCR of a 6 kb plasmid using mix polymerase. I used the 5' phosphorylated primers for blunt end ligation. BUt after ligation i am not gettion any colonies. My ligase, plates, competent cells are good and working for other cloning work. But this is not working . can anybody expalin the cause of failure. If any one has suffered such problem in the past and overcome it by some methods. please share your ideas with me.
sunil[size="5"][/size]
I have done reverse PCR of a 6 kb plasmid using mix polymerase. I used the 5' phosphorylated primers for blunt end ligation. BUt after ligation i am not gettion any colonies. My ligase, plates, competent cells are good and working for other cloning work. But this is not working . can anybody expalin the cause of failure. If any one has suffered such problem in the past and overcome it by some methods. please share your ideas with me.
sunil[size="5"][/size]
...you wanted to yell again, why do you need to do so anyway ? we can see what you write without having to use font 5...
there are posts like yours in the forums, just digg a bit... Just think that cloning a large fragment is tricky, you should use electrocompetent cells, and I have given a link to some kits from epicentre biotechnologies... in some other post but I don't remember where... it makes use of a fairly large plasmid (8Kb instead of common 2-4Kb)...
-ph3no-