ligation of 2 linear sticky fragments - (Jun/13/2005 )
Hi everyone.I wanted to ligate two linear fragments obtained after RT-PCR.Idigested them with the same enzyme to create sticky ends .I started with 1 microgram 0f PCR products for digestion but after ligation when I checked on gel I only saw a smear from top to bottom of the gel.Could you suggest me what might be the reason? Should I start with the more concentration of PCR products or should I change the ratio .
Not sure about this one, it sounds like your DNA is being degraded. After digestion and clean up do you check your product on a gel before ligating. It seems to me that somewhere along the line something is chewing up the DNA.
After ligation you would expect a ladder of DNA products, this should be discrete and increase in size by the size of either of your inserts. How big are the fragments you are cloning?
One fragment is 820 bp and another fragment is 538 bp length.Both have Nhe1 restriction sites.Actually I have to ligate total 5 fragments one by one and finally clone them into a vector. All fragments are RT-PCR products and will have sticky ends after restriction digestion. Is it possible to ligate them altogether in the same ligation reaction because all are having sticky ends.
I'm still worried about your DNA smear, which makes me think that something is degrading your DNA, I would try fresh stocks of restriction enzyme, ligase buffer and ligase, it seems as though you are getting DNAse contamination somewhere.
As far as ligatging all five fragments at once, technically it can be done, however, if they all have the same restriction site you will have no control with order of ligation or composition. You will end up spending more of your time trying to find a construct with one of each construct then doing suquential ligation reactions.
Thanks for the reply.I will try new stocks of enzymes and buffers.