August 25th, 2010
1. Run gel to verify PCR made on August 24th.
￼Lanes: 1) Green ladder; 2) pSB3K3 RBS-GFP FWD-J23101 REV (reaction 1); 3) pSB3K3 RBS-GFP FWD-J23101 REV (reaction 2); 4) Preffix FWD-Suffix REV; 5) Suffix FWD-J23101 REV; 6) RBS-GFP FWD-Suffix REV (Tube 5).
- This PCR was succesful!! (at least reaction 1)
2. Purify PCR product from pSB3K3 RBS-GFP FWD-J23101 REV (reaction 2) using the High Pure PCR Product Purification Kit Roche.
3. Make restriction to purified PCR product with SpeI-XbaI.
- Double restriction methods:
- DNA -> 5ul
- Buffer 2 -> 2ul (10% of total volume)
- BSA (required by PstI) -> 1ul
- SpeI -> 1ul
- XbaI -> 1ul
- HPLC -> 10ul (to complete total volume of 20ul)
- Incubate at 37ºC 3.5 hrs.
4. Make ligation of BBa_I20260 with the new RBS.
- Ligation methods (Total volume 20ul):
- DNA -> 5ul of p30-MinBP
- Buffer for T4 DNA ligase 5X -> 4ul (Final concentration 20%)
- T4 DNA ligase -> 1ul
- HPLC -> Complete total volume (20ul)
- Incubate overnight at 16ºC
- Mix well (vortex) buffer and reaction tubes.