inverse PCR molecular bilogy
Posted 24 June 2009 - 05:17 AM
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
Posted 24 June 2009 - 04:17 PM
Posted 25 June 2009 - 02:43 AM
Anyway, you could try to re-design your primers
do a gradient on the annealing temperature.
Posted 28 June 2009 - 11:50 AM
* 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.