October 20th, 2010
1. Run gel to verify results from PCR made on October 15th.
Lanes: 1,12) Green ladder; 2-4) pSB4A5 + MinBP + ΔRBS + GFP E004 Colonies 1-3, 5-6) pSB4A5 + MinBP + ΔRBS + GFP E004 Colony 6 10; 7-10) pSB4A5 + MinBP + ΔRBS + GFP E004 Colonies 7-10; 11) BBa_I51020 amplified with Preff. FWD-Suff. REV.
2. Inactivate enzyme from ligation made on October 15th for 20 min at 65ºC.
3. Purify PCR product from colony 7, purification was done with the High Pure PCR product Kit Roche.
- This product contains the construction: MinBP + ΔRBS + GFP E004.
4. Make restriction with EcoRI-PstI to purified PCR product (MinBP + ΔRBS + GFP E004).
- EcoRI-PstI double restriction methods:
- DNA -> 5 ul
- Buffer 2 -> 2 ul (10% of total volume)
- BSA -> 1 ul
- EcoRI -> 1 ul
- PstI -> 1 ul
- HPLC -> 10 ul (to complete total volume of 20ul)
- Incubate at 37º C for 4 hours.
5. Transform the following ligations:
- pSB3K3 + J23101 with ΔRBS + GFP E004 (20ul)
into DH5-α competent cells, transform also BBa_I51020 (p19) (5 ul DNA) as control.
- Unfreeze competent cells (keep on ice).
- Add exogenous DNA to 50ul of compentent cells.
- Incubate 20 min on ice (during this time the DNA and the cells interact to achieve transformation).
- Incubate 1 min at 42ºC (this step increases transformation efficiency, it allows membrane motitlity and close the Ca channel).
- Incubate 5 min on ice.
- Transfer the cells to a tube with 1ml of LB medium
- Incubate the cells in the LB medium for 1hr at 37ºC and shaking (during this period the cells recover their metabolic activity, replicate and synthesize the proteins encoded in the material just inserted).
- Transfer the LB medium with the cells to an eppendorf tube, centrifugue 1 min at 13000rpm.
- Discard the supernatant keeping just ~50ul of medium and resuspend the pellet in this volume.
- Plate the resuspended cells on dishes with the correspondent antibiotic. Add ~10 pearls and shake 1-2 min.
- Incubate the plates at 37ºC overnight.
6. Inactivate restriction enzyme for 20 min at 80º C.
7. Make ligation:
- pSB1C3 (3ul) + [MinBP + ΔRBS + GFP E004] (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.