Revision as of 08:17, 25 October 2010 by RussellDurrett (Talk | contribs)

While the 3A assembly method is well suited for stepwise building of devices, larger complex constructs can take time to make. Being the impatient New Yorkers we are, as soon as we came up with the constructs for our project we had to have them immediately. Because of this, we began exploring other methods to piece together our system.

We decided to use the Overlap Assembly Method most famously published in this JCVI paper:

Gibson et al, Nature Methods 6: 343-345

This method allows researchers to assemble complex systems of many parts all at once rather than in a stepwise fashion. The downside of this system is that the parts to be assembled must overlap one another in their sequence, so you must order DNA oligos to PCR constructs into overlapping forms. To help us out with this, Russell programmed an overlap oligo maker that did much of the work for us - check it out by clicking this link:

After getting the oligos in the lab we combined equimolar amounts of each part in a single reaction and added ExoIII, Phusion polymerase and Taq Ligase. We opted for a 2-step thermocycled reaction method for simplicity and cost-effectiveness and the relative ease of making a reaction mix. Using the reaction mix suggested in the Gibson et. al. paper, we designed our own simplified version by using the Phusion and Ligase prepared buffers. We were able to get successful reactions from this mix:


We kept this reaction mix at 50C for 3 minutes to let the ExoIII digest our DNA, then heat inactivated it by keeping it at 75C for 20 minutes. When cooled down to 60C, the now single-stranded parts annealed to one another and the Phusion polymerase and Taq ligase filled in and closed the gaps. The yields were too low to clone directly from this reaction so instead we PCRed full constructs using BB prefix and suffix reverse complement primers and high fidelity polymerase.