5 replies to this topic

[tck1]

I am trying to ligate an insert (1.5kb) cut with  BamH1/HindIII into a similarly cut standard vector (4Kb). I have tried different ligases, different temeratures, volume excluders such as PEG and even a very expensive rapid DNA ligation kit, but I still cannot get the insert and vector to ligate. Does anyone have suggestions. Thanks.  

[Rybak]

May be cloned fragment is toxic for Your bacteria.
If you have primers on the both sides (for ex. before BamHI and after HindIII) You can make PCR. If after PCR you see line of DNA and size is similar to you cloned fragment and after transformation you have no clones then you have not problem with ligation reaction. The matter in toxity of cloned fragment.

[hello]

how about your ligation?
i have the same problem,can you help me ?

[anonymous]

Are you sure your vector has no problem?  I sugest you should purify your vector again.

[yaochunwu]

purified vector and  insert are necessary now;

increase the insert /vector ratio

[yaochunwu]

incrase DNA ammount in transformation

increase insert/vector ratio

purify both insert and vector before ligation