Maybe I'm overcomplicating this, but I'm having some issues with a construct.

So, I have three plasmids containing a total of eight DNA sequences that I need to fuse into one linear sequence. I plan to fuse the sequences using Gibson assembly. However, before I can fuse them, I need to amplify (PCR) them out of their plasmids.

I would like to use as few primers as possible, so my question is this; can I use the Gibson assembly primers to amplify them out of the plasmids and add the overlap sequences separately, then fuse them all together in a separate reaction without primers? Would the overlap sequences already added to each sequence separately be enough to act as primers for assembly? Even better (but I doubt it would work), could I amplify them out of their plasmids and fuse them in the same reaction?

If both these scenarios are unrealistic, I can still just order another round of primers to extract the sequences from their plasmids, but I would like to just use the primers I have if I can.

  • $\begingroup$ It's a little unclear what you mean by 'add the overlap sequences seperately' Normal gibson primers look like this: 5'Overlap sequence- Binding region-3' You can use these to PCR our the fragments without any issue. I've added 5' extensions of 60bp without problems. Gibson assembly normally occurs without any primers as you purify the PCR fragments first. Your reaction will be very inefficient with 8 fragments, gibson really peters off at 6 put you can be lucky sometimes... $\endgroup$ – mimat Oct 6 '16 at 16:15
  • $\begingroup$ @mimat - I am mostly wondering if the plasmids will get in the way of my assembly at all. After a little discussion with some colleagues, I have determined that it will probably be fine as long as I add the overlap sequences first with their amplification primers, then fuse them in a different PCR reaction (in Gibson assembly, I have always just added my sequences and the overlap primers to the same PCR reaction). I forgot to mention in my original question that I will actually be splitting the fusion of all eight sequences into two or four different reactions. Thank you for clarifying. $\endgroup$ – CDB Oct 6 '16 at 18:33

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Browse other questions tagged or ask your own question.