September 15th, 2010
1. Inactivate restriction enzyme from reactions prepared on September 14th, 20 min at 80º C.
2. Run gel to check restrictions and PCR made on September 14th.
Lanes: 1,8) Green ladder; 2) pSB3K3 + J23101 SpeI-PstI restriction; 3) Δ RBS + GFP E0040 XbaI-PstI restriction; 4) BBa_I20260 Min-BP REV-Suffix FWD PCR product (tube 7); 5) BBa_I20260 Min-BP REV-Suffix FWD PCR product (tube 8); 6-7) Claudia’s samples.
3. Purify PCR product of BBa_I20260 Min-BP REV-Suffix FWD using the High Pure PCR Product Purification Kit Roche.
4. Make restrictions to pure PCR product, plasmid pSB3K3 + Minimum Blue Promoter with SpeI-PstI.
- SpeI-PstI double restriction methods:
- DNA -> 15 ul
- Buffer 2 -> 4 ul (10% of total volume)
- BSA -> 1 ul
- SpeI -> 2 ul
- PstI -> 2 ul
- HPLC -> 16 ul (to complete total volume of 20ul)
- Incubate at 37º C for 2 hrs.
5. Inactivate enzyme from pSB3K3-Min. Blue Promoter SpeI-PstI restriction, 20 min. at 80 ºC.
6. Make the following ligations:
- pSB3K3-J23101 SpeI-PstI (3ul) with Δ RBS-GFP E0040 XbaI-PstI (5ul).
- pSB3K3-Min. Blue Promoter SpeI-PstI (2ul) with Δ RBS-GFP E0040 XbaI-PstI (5ul).
- Ligation methods (Total volume 20ul):
- DNA -> Volume of each part to be ligated is indicated above.
- 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, unfreeze buffer on ice.