dear all,

I am trying to ligate 3 pcr fragments together. The middle fragment has different RE sites on the ends and they are compatible to one or the other remaining fragments. The pcr reaction creates clean bands.

https://documents.epfl.ch/users/o/oz/ozdemi...ublic/11_07.tif

Also, after digestion with REs (and Antarctic Phosphatase treatment for the middle fragment) displays the same clean bands on agarose gel. However after an attempt to ligate them and hoping to get the right sized fragment out of the gel - all I got was pretty much a smear. (ligation rxn in 20ul ~200ng total DNA 16C o/n T4 dna ligase - smears are for 1:1:1 1:2:1 and 1:3:1 mix of fragments)

https://documents.epfl.ch/users/o/oz/ozdemi...ublic/11_04.tif

Is there any suggestions that can help me fix it (well, even understanding would be nice!)?
or was it too much to ask for to get such a ligation?

Thanks!

Hi there!

Happy Friday!

I can't view your links because it's asking for a username and password
Quick question - are you trying to ligate these 3 PCR fragments into a vector or just stick them together? Is it possible that there are many ligation options available, hence why you are seeing a smear and not one discrete band?

Clare