Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Biology

cloning in pET 30 B - (Dec/13/2009 )

Dear friends
Im trying to clone a 4.2 kb PCR product in the expression vector pET30 b. First i tried T/A cloning but i could not get the clone. i dont know why, i assume a 4 kb insert is too big for T/A cloning
Next i double digested the pcr amplified product with Nde1 and Xho 1 enzymes. i did overnight digestion before ordering the primers, i did add extra nucleotides at the 5-end of the primer before the restriction site.. But still i didnt get any spite of all correct conditions ..ligation, transformation etc
meanwhile my PCR amplicon is getting over, but i cant do PCR all the time since the kit with the high fidelity PCR enzyme is getting over..
what can i do..
pls help


Hi chn, whenever I hv had trouble wid cloning, it has always been coz of incomplete digestion by the REs. Maybe u first should figure out whether the enzymes are working fine (if u haven't already done it). n also u could confirm that the RE sites that u hv in ur product are fine, no mutations (even though u use high fidelity PCR enzyme).....was just wondering if the primer by mistake has a wrong sequence....

Also, when u do the transformation, do u get religated vector or no colonies at all???