Progress! The big fragment-assembling PCR is running!

The PCR amplification of the SpcR fragment worked fine yesterday (after I spent an hour staring at (forward and reverse and complement and reverse-complement) primer sequences to reassure myself that I'd ordered the correct ones).  And today I did a column cleanup and a gel purification.  And now I'm running the PCR reaction that I hope will assemble the complete DNA fragment.

The part I'm doing now is shown below.  You can see the whole plan in this post.

When I was getting ready to do the PCR I realized that first I needed to think in more detail about the events.  So here they are, in point form:

1. Most of the green strands will just reanneal to their complements.  Probably many of the red and blue strands do too.  This is an unwanted process that reduces the availability of strands for the desired annealings and elongations. In normal PCR this is hindered by the very high concentrations of the primers, but here we only have the F and R primers.
2. Primer F anneals to the lower red strand and primes synthesis of the upper red strand.  At the same time, primer R anneals to the upper blue strand and primes synthesis of the lower blue strand.  Both of these new strands accumulate linearly, not exponentially.  This provides more of the appropriate strands red and blue for STEP 4.
3. STEP 4 above: Sometimes the upper red strand anneals to the lower green strand (by their 17 bp of complementarity), and elongation in both directions produces hybrid upper and lower (red + green) strands.  Similarly, sometimes the upper green strand anneals to the lower blue strand (by their 16 bp of complementarity), and elongation in both directions produces hybrid upper and lower (green +_ blue) strands.  All these strands also accumulate linearly, not exponentially.  They provide the effective strands for STEP 5.
4. STEP 5 above: Sometimes the hybrid strands from STEP 4 anneal to each other instead of to their complements (by their 1260 bp of complementarity). Elongation in both directions produces the full-length strands.
5. STEP 6 above: Now primers F and R can anneal to the full-length strands, leading to exponential amplification of this desired product. 

Miscellaneous thoughts:

• I don't know how much template is appropriate, so I'm trying two concentrations, one 100-fold lower than the other.
• The rate-limiting step is STEP 4, the annealing of the red and green strands by their 17 bp overlap, and of the green and blue strands by their 16 bp overlap.  I'm using a fairly low annealing temperature to facilitate this.  If I don't get any product I'll try lowering it further.
• If I hadn't mixed the red and blue fragments for purification I might have set up partial reactions (red + green and blue + green), to make it easier to monitor progress.  If the all-in-one reaction doesn't work I'll probably make some more of each fragment and purify them separately so I can troubleshoot in separate reactions.