LONG OLIGOS (100+ BASES)

────────────────────────────────────────────────────────
CHEMICAL SYNTHESIS: HIGH-LEVERAGE CHANGES (DO THESE FIRST)
────────────────────────────────────────────────────────
1) Switch to dGibu phosphoramidite (NOT dGdmf)
2) Use 3% DCA (instead of TCA) for deblocking
3) Double-couple AFTER base 100 (earlier if RNA)
   • Add a second coupling (or extra reps/time) after ~100 bases to maintain coupling efficiency.
4) Use larger-pore CPG for long sequences
   • Use 3000Å CPG (or higher if available) for 100+ base oligos.
   • If keeping oligos <100 bases, 2000Å CPG is typically fine.
5) Optional: synthesize onto non-porous polystyrene beads (if available)
   • Advantage: not limited by pore steric hindrance.
   • Tradeoff: lower surface area vs porous supports → lower yield.
6) Otherwise: keep standard protocols
   • Don’t change everything at once. Make the changes above, then reassess.

────────────────────────────────────────────────────────
SCALE / LOADING REALITY (IMPORTANT)
────────────────────────────────────────────────────────
Long-oligo success is scale-dependent.
• Smaller scales can behave better at long lengths (less crowding; more effective equivalents).
• Higher loading densities / crowding reduce effective coupling as length increases.

If long oligos are failing at high scale, test a smaller scale and/or lower loading density.

────────────────────────────────────────────────────────
PRACTICAL RULE: DON’T FORCE ONE ULTRA-LONG OLIGO IF YOU DON’T HAVE TO
────────────────────────────────────────────────────────
If your end goal is a “gene-length” construct, it’s often more reliable to:
• synthesize shorter oligos (that your system can make well),
then
• assemble them into longer DNA by ligation, PCR tiling and cloning.

This keeps the chemistry in the zone where it performs best, while still letting you build long constructs.

────────────────────────────────────────────────────────
PCR TILING / OLIGO ASSEMBLY (HIGH-LEVEL WORKFLOW)
────────────────────────────────────────────────────────
Goal: stitch shorter overlapping oligos into a long dsDNA fragment using overlap-extension PCR.

A) DESIGN
• Break the target sequence into tiled oligos with overlaps.
  Typical overlap: ~20–30 bp (enough to anneal specifically).
• Keep overlaps consistent in melting behavior where possible.
• Avoid strong hairpins/repeats in overlaps (they create mis-annealing).
• If the construct is very long, plan a hierarchical build:
  - assemble sub-fragments first,
  - then assemble sub-fragments into the full-length product.

B) OLIGO PREP
• Use high-quality oligos (purification as appropriate for length/complexity).
• Mix oligos in equal molar ratios for assembly (unequal ratios leave leftovers and increase byproducts).

C) ASSEMBLY PCR (TWO-STAGE CONCEPT)
• Stage 1: “stitching” (overlap extension)
  - Run cycles that allow overlaps to anneal and extend without relying heavily on outer primers.
• Stage 2: “amplification”
  - Use outer primers to amplify the full-length assembled product.

Use a high-fidelity polymerase and minimize unnecessary cycling to reduce errors.

D) VERIFY
• Check product size by gel.
• If multiple bands: optimize overlap design, assembly grouping (sub-fragments), and cycling conditions.

────────────────────────────────────────────────────────
CLONING TO CREATE A STABLE “GENE” (STANDARD PRACTICE)
────────────────────────────────────────────────────────
Once you have the assembled fragment:

1) CLONE INTO A VECTOR
• Use a cloning method compatible with your fragment ends and your lab’s standard workflow
  (e.g., homology-based assembly or restriction/ligation approaches).

2) TRANSFORM / SCREEN
• Screen multiple colonies (PCR screen or restriction digest).

3) SEQUENCE CONFIRM
• Sequence the insert to confirm correctness (critical for long constructs).

4) SCALE / STORE
• A confirmed plasmid clone becomes the stable source for that “gene” going forward.

────────────────────────────────────────────────────────
TROUBLESHOOTING (COMMON FAILURE MODES)
────────────────────────────────────────────────────────
If assembly fails or gives smeared/multiple bands:
• Reduce the number of oligos assembled in one reaction (build sub-fragments first).
• Increase overlap robustness (length/sequence uniqueness).
• Rebalance oligo molar ratios (equal molar is the default).
• Watch for hairpins/repeats in overlaps.
• Ensure oligos are fully resuspended and quantified consistently.

If you’re not getting enough full-length:
• Don’t just “run more cycles.” Fix assembly design or build hierarchy first.
• Consider double coupling earlier and/or larger-pore supports for the longest oligos. 

────────────────────────────────────────────────────────
BOTTOM LINE
────────────────────────────────────────────────────────
For 100+ nt oligos:
• Use the chemical synthesis levers first (dGibu, 3% DCA, double coupling after 100, larger-pore support)
For “gene-length” constructs:
• Build from shorter, reliable oligos using PCR tiling + cloning for stability and verification.