weird problem in cloning!! - (Apr/15/2012 )

i have been facing this problem over a period now….
i have a gene in a plasmid (1 for the namesake) and need to transfer into another (let be plasmid 2)….so after digesting the gene from plasmid 1 and gel extracting it….i digested the plasmid 2 and set up a ligation.After ligation i transformed the mix in DH5alpha and grew them for screening.
after a miniprep….i got my product in a pcr but after an RD….no insert is released…moreover i get double bands in my digestion mix….(one corresponding to my digested vector…and other higher than that....that somewhat looks like a nicked form...but i am not sure about that….)


has anybody faced this problem…or any suggestion….(i did try troubleshooting almost all parameters and vary all
conditions…but the problem persists..)


RD is done with nde1 and kpn1(i checked for enzyme activity…they work fine)
i also checked with the cultures ...they are also fine....

Is there any restriction sites (what you have chosen for digestion) in the enzyme sequence or in the vector other than ligation site.

no...the gene has the restriction sites on the flanks....and for the target plasmid...they are in the mcs....i have cross checked..that there are no recognition sequences for the enzymes...in hese two entities(insert and plasmid)....

Colony pcr has problems when used to evaluate cloning results when the primers are both on the insert. I'd recommend using one or two primers on the destination plasmid to amplify, and then checking the length of the product. Carryover of the insert DNA which is not cloned onto the plate is common, an can be amplified during colony pcr.

@phage434.....thanks for the suggestion....but...at this point of time....i am not concerned about the insert part...but a greater concern are the different bands that appear even after a digestion of even 3-4 hours.If i can get through this...i am quite sure...the insert part can also be taken care of...