Hello everyone,
I've been trying to perform a ligation for the last couple of weeks and I just get empty plasmids.
The insert is pool of fragments amplified by PCR with an average length of 3kb and cut with the same restriction enzyme on both ends. I am trying to insert this pool of fragments in a vector that is 5,5kb long and is cut by the same enzyme. Not the best option for ligation but this is something I cannot change at the moment.
I am aware that religation of empty vectors is quite probable here so I performed double dephosphorylation of the vector (FastAP and Antartic). I also tried different ratios of vector: insert (1:3, 1:1, 3:1) and I keep getting a similar number of colonies in control and ligation plates.
I've also made some colonies PCR and none of the 30/20 colonies picked by plate showed an insert band. Every clon was an empty plasmid. I am considering moving the protocol to a vector that allows white/blue colonies screening but still, efficiency is really low if there's any....
Does anyone have any advice or suggestion for the ligation? Am I overlooking or missing something?
Thank you very much in advance to you all
Jorge
you need a selection, and use smaller pUC type vector first .
what is your transformation efficiency ? what is your cut and ligation control ? do your ligase works ? have you cleaned PCR products ? it helps sometimes. are your restriction sites at the end or you have some additional bases?
You can try cloning by using a restriction site on the vector that is compatible, but not recleavable after ligation, with the restriction site on the PCR bands. That way, you can recleave empty vector molecules post-ligation (or include both restriction enzymes in the ligation mixture and cycle between 16 and 37*C, a la Golden Gate cloning) and the recombinant clone/empty vector ratio will increase.
By the way, did you include a 'stuffer' of 3-4 bp at the 5' end of the primers before the restriction sites? Also, have you checked that the ligation actually works? ATP in most commercially available ligation buffers hydrolyzes quite rapidly; maybe you could supplement the ligation with fresh buffered ATP.
Hope it helps!
2 things to watch out for. If your restriction enzyme leaves a 3' overhang then dephosphorylation may be very inefficient, try doing it at 55oC. Also, if your PCR primers have the restriction site at the very end, they may cut poorly. Try making primers that have a bit more sequence before the actual 6 bp restriction site. Add some random As and Ts for example.
Try using a different protocol. I've easily cloned 7.5 kb fragments with a "Topo-TA cloning kit". No need to do any restriction digests or dephosphorylation treatments. As long as your PCR polymerase leaves an A overhang on the PCR product, you directly add the PCR product to the plasmid vector. The kit is a bit more expensive than most, but it works so well it's worth the cost.
Katie A Burnette good idea but its expensive a little .... topo usually works very well :)
he can do terminal transferase cohesive ends too; A for PCR and T tail for vector anneal add klenow or other polymerase to fill the gaps and ligate... TT is also not cheap but maybe its in on site with other enzymes
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