Cargo ordering and pool preparation
Protocol 3 of 7. Comes after Primer plates. Next is Cargo creation.
Order the synthesised material this library is built from, and put it away ready for the bench.
Leaves you with
- oligo pool the pool resuspended
Materials
| Name | Supplier | Catalogue | Storage | Per run | Note |
|---|---|---|---|---|---|
| Oligo pool | -20 °C | 131 oligos, every one 350 nt, resuspended to 20 ng/µL | ordered from pool.tsv, which says which block each oligo is a piece of | ||
| NEBridge Golden Gate Assembly Kit, BsmBI-v2 | NEB | E1602 | -20 °C | one assembly per block | its mix carries the BsmBI that cuts every oligo back to its own fragment, which this method reserves, and the T4 DNA Ligase that joins them |
Order and store
Expected result
- One pool, 131 oligos, every one 350 nt and no length spread, which is what the padding is for.
Troubleshooting
- The vendor bins the pool by length
- Every oligo is padded to one length, so the spread is zero. A vendor asking for a different length wants this run's oligo length changed and the design run again.
Notes
- pool.tsv names each oligo's block, which piece of it that is, and the primers that pull it out, so an oligo is traceable to its protein.
- 131 oligos is the fewest the length budget allows for.
Caution: Spin the tube down before taking the cap off.
Expected result
- One tube of pool in solution at 20 ng/µL or above, with nothing left undissolved on the wall of the tube.
Notes
- Dividing by 20 is what the floor of at least 20 ng/µL comes to, and the amplification then pipettes 1 µL of 20 ng/µL.[1]
Sources
- Twist Bioscience, Twist Oligo Pools Amplification Protocol · DOC-4060 REV 1.0 · held under reference_docs/ · docs/research/oligo-pool-pcr-cycles.md cited at Before You Begin