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problem with digestion and ligation of BgI II and kpnI RE - (Feb/26/2006 )

unsure.gif i am having problem to ligate my digested pcr product, which have BgI II and kpnI cut sites, into pET 28. interestingly, among the numerous trials that i performed i was able to have one succesful ligation. but i am unable to reproduce the experiment with different DNA with the same conserved endunucleases or cut sites, BgI II and kpnI . i appreciate any suggestions, thank you


i want to know exactly what u r doing, i mean after pcr what u r doing u r digesting the pcr fragemnt with ur restr enzys then ur eluting the fragment from u r digested sample and then u r keeping for ligation into a vector (which is already digested by same enzymes).

if yes how u know that double digestion is perfectly done?
and after ligation what u r doing how u know it is not ligated?
if it the problem with Restrn enzymes u do digestion of vector and conform that it is working fine,and if it is happening with ligation u do +ve control means u elute the fragmnt from the vector after digestion and keep it as for +ve contrl for ligation? and also atp in a buffer will exhaust if the buffer is multiple thawed then use some atp in that buffer or use fresh buffer or u try at different temparutes and with diffrnt enzymes also.