July 2nd, 2010
1. Make XbaI-PstI double restriction to GFP BBa_K145015 (74 min) extracted by PCR.
- Double restriction methods XbaI and PstI (DNA 10 ul, Total volume 30 ul):
- DNA -> 10 ul
- Buffer 3 -> 3 ul (10% of total volume)
- BSA (required by SpeI) -> 1 ul
- PstI -> 1.5 ul
- XbaI -> 1.5 ul
- HPLC -> 13 ul (to complete total volume of 30ul)
- Incubate at 37ºC overnight
2. Inactivate enzyme from XbaI-PstI double restriction to GFP E0240, made on July 1st. 80ºC for 20 min.
3. Dephosphate p30-MinBP PstI-SpeI, this plasmid will be ligated to GFP E0240 XbaI-PstI. Incubate 20 min at 37ºC and 10 min at 65ºC:
- DNA -> 15ul
- Buffer -> 3ul
- Phophatase -> 1ul
- HPLC -> 11ul
- Total volume: 30ul
4. Inactivate enzyme from XbaI-PstI double restriction to GFP BBa_K145015 (74 min), 80ºC for 20 min.
5. Run gel to verify parts to be ligated:
- Dephosphated p30-MinBP SpeI-PstI
- GFP E0240 XbaI-PstI
- Dephosphated backbone pSB3K3 EcoRI-PstI
- Blue Promoter (extracted by PCR) EcoRI-SpeI
- GFP BBa_K145015 (74 min) XbaI-PstI
- Dephosphated p18 XbaI-PstI
GEL WAS A TOTAL FAIL, but I’ll even do ligations.
6. Make ligations:
- 1. Dephosphated p30-MinBP SpeI-PstI (3ul) + GFP E0240 XbaI-PstI (6ul)
- 4. Dephosphated backbone plasmid pSB3K3 EcoRI-PstI restriction (3ul) + Blue Promoter EcoRI-SpeI restriction (5ul) + GFP BBa_K145015 XbaI-PstI restriction (5ul)
- 5. Dephosphated plasmid 18 XbaI-PstI restriction (3ul) + GFP BBa_K145015 XbaI-PstI restriction (5ul)
- Ligation methods (Total volume 20ul):
- DNA -> Depends on concentration
- Buffer for T4 DNA ligase 10X -> 2ul (Final concentration 10%)
- T4 DNA ligase -> 1ul
- HPLC -> Complete total volume (20ul)
- Incubate overnight at 16ºC
- Mix well (vortex) buffer and reaction tubes.
7. Replate transformation Backbone pSB3K3 + Blue Promoter + GFP E0240, this transformation was done on June 29th.