High transformation background in negative control - (Aug/30/2006 )
i dont know what is happening this time with cloning. i tried to do it two times and alsways get many many colonies on the negative control plate... i cut out of the gel, purify etc....i even cut with some enzyme that cuts inside MCS, to get rid of self ligated stuff....nothing works... and i dont think im gettting the insert there.. .....can a vector be resistant to ligation??
If you get many colonies on the negative control plate, there is something wrong with your selection system.
Anitbiotic? - too little, or too old, or mistreated antibiotics.
Or, the host organism is resistant to the selection.
Make fresh plates! maybe increase amount of selection agent.
Use a fresh solution.
Do not over heat the selection agent
well... basically, you can also dephosphorylate, but that's an easy answer...
I'm also stuck with ligations and i "play" with vector insert ratios. so many ligations / plates...
And i screen only if the "positive plate" has 3-4times more colonies than neg one.
I have background, but the relative proportion of the positive/neg gives mechances to get the good one.
I quite agree with fred.
I might increase the vector conc. to 100ng and have 5-10 fold more of insert. It might b unconventional but it works for difficult ligations. I would even pick colonies if I have 2x more colonies in the positive than negative as I need only a single right colony.
good Luck !!!
more info would be nice. What have you done? It is hard to spot any problemsin the protocol you are following with your current explination.
Something is obviously going wrong here. IS your CIP, ligase buffer and ligase still good? All three will go bad after repeated freeze thaw cycles. Ligase goes the fastest, and seemed to go bad even at a constant minus 20.
P.S Don't leave out the gel purification step on the vector. You get denatured vector coming through. Ie vector DNA that can not be cut but can transform rather nicely.
P.S.S: If the vector is sufficiently big it can be very very hard to ligate. Also if any of the DNA component have regions of high AT contant then you can have problems with tertiary structure.
thanx a lot everyone, i have done two ligations at the same time, using samely purified vector but different PCR inserts. one worked and the other not, so i guess it must be the insert:vector ratio that mattered and i will increase it in the case of the failed ligation. but my confusion is plate that has so much negative clones! i cut out of the gel and dont do CIP, but i suppose most of the clones should be double digested
Unless you have 100.000% digestion, you are looking at potential heartache with your ligations, sorry to say.
I remember hearing a Nobel prizewinner say that the only way to be sure is to do the experiment. Seeing as how 100% guaranteed digestion not likely at all in this lifetime, I'd try a bit of CIP.
Think of it this way: you are an intelligent sentient being. The DNA with which you struggle is not. You will win!
Perneseblue, could you please further explain this comment in relation to successful cloning (or anyone else who might know).
If you ever have to make a multi component plasmid which incorporates a 5kb segment composed of >75% AT, do not try to add that fragment via 3 way ligation. Go via a 2 way ligation.
AT rich DNA is bent.
I am guessing it adds topological strain to the plasmid. And I believe E.coli doesn't like it either, especially when said fragment becomes larger.