inverse PCR molecular bilogy - (Jun/24/2009 )
i have ligated my gDNA for circulation of different fragments after enzyme digest.i want to clone gene by using inverse pcr i have partial sequences for designing primers.but after inverse pcr i got many bands
i dont' why every thing was fine and i took alot of care too
i need your help
Optimise your PCR- magnesium and temperature gradients will help get empirical conditions at which your PCR results in only one or two of bands. Theoretical Tm and other reaction conditions always need to be tested to see if they work in real life.
i did IPCR before too, never seems to work on my hand. really feel like doing it again sometimes haha.
Anyway, you could try to re-design your primers
do a gradient on the annealing temperature.
If you cut and religate using a single enzyme, then multiple copies of the same fragment can assemble, rather than circularization. This can happen in two ways. You get at least these assemblies:
* circularization of a single fragment
* assembly of two distinct cut fragment oriented head-tail (reconstruct prior sequence)
* assembly of two distinct fragments head-head
* assembly of two distinct fragments tail-tail
The first one gives the result you are looking for. The last two give PCR fragments when amplified using a single primer (a different one in each case). You can bias the ligation reaction in favor of your desired result by ligating at low DNA concentration, such that the local concentration of same-molecule end is high compared to the local concentration of different-molecule end.
Depending on the length of your fragment, this sweet spot is around 1 ng/ul or so. You could do serial dilutions of your cut DNA, followd by ligtation and PCR. The effective direction is likely to be low concentrations.