Team:UCLA/Notebook/Spider Silk Genetics/3 June 2015
6/3/2015
Oligo Pre-Treatment
- We recently received the initiator, terminator, and capping oligos.
- We ultimately decided to order doubly biotinylated initiator since it could provide higher yield.
- Each oligo was made into 100 uM stock.
- According to the ICA Paper (Briggs et al., 2012) we prepared the oligos as follows:
- Double stranded oligos were mixed together with 100 uM of each.
- Single stranded oligos were diluted to 5 uM each.
- All oligos were treated heat treated at 95 C for 1 min, the ramped down to 25 C at 0.1 C/s.
- After heat treatment, the oligos are called working oligos.
ICA oligo characterization
- We tested each BsaI digested fragment with each capping oligo, and tested the initiator and terminator with AB and CA respectively.
- We used 50 ng of each fragment generated yesterday.
- Oligos were used as follows (copied from (Briggs et al.):
- Initiator and terminator: 1 uL of 50 nM working solution
- Caps: 1 uL of 5 uM working solution
- 0.5 uL of T7 Ligase.
- ddH2O to 10 uL.
Results
- Cast 2.5% TAE gel, used 2 uL of NEB 50 bp ladder.
Fig. 1 Characterization of initiator, terminator, capping oligos with MasP AB, BC, CA. The first lane is the negative control. The 102 bp band corresponds to unligated monomer size. The initiator is 59 bp, the terminator is 39 bp, and the caps are 20 bp. The 122 bp bands correspond to the capping oligos ligated to the monomer.
- We showed that the caps work as intended and would be usable in ICA.
- We expected the following combinations to ligate.
- AB + Initiator (inconclusive)
- AB + B-cap
- BC + C-cap
- CA + A-cap
- CA + Terminator (inconclusive)
- There was an insufficient amount initiator and terminator in the reaction, so no bands. But the sticky ends should work as expected.
- CA weakly ligates with C-cap, which is shown by the double bands present. This result was not expected, and may be due to non-specific ligation.