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Team:Groningen/1 September 2010
From 2010.igem.org
Assembly of biobricks in pSB1C3-backbone for submission to parts registry by Maarten
O/N cultures of E. coli top 10::pSB1C3-Jo4450 and E. coli top 10::pMASK-C/pMASK-DC (acquired from Mr. Gene) were miniprepped with a Bioké miniprep kit and restrictions on pSB1C3-RFP and pMASK-C, p-MASK-DC with XbaI and PstI were performed. E, H and Sortase fragments were already restricted with XbaI and PstI by G. van Heel. Mixtures were:
| 1,0 µL pMASK-C | 1,4 µL pMASK-DC | 1,5 µL pSB1C3 |
| 2,0 µL Tango buffer (10x) | 2,0 µL Tango buffer (10x) | 2,0 µL Tango buffer (10x) |
| 0,5 µL XbaI | 0,5 µL XbaI | 0,5 µL XbaI |
| 0,5 µL PstI | 0,5 µL PstI | 0,5 µL PstI |
| 15,5 µL MQ | 15,1 µL MQ | 15,0 µL MQ |
Incubation for 1 hour @ 37°C.
Fragments C, DC, E, H and Sortase were ligated into pSB1C3 vector:
| 10 µL insert |
| 7,5 µL vector |
| 5,0 µL T4 Ligase |
| 2,5 µL T4 Ligase buffer |
Incubation for 1 hour @ RT.
Ligates (10µL) were transformed to E. coli and grown on LB agar plates containing 15 µg/mL Chloramphenicol. For negative control 10 µL MQ was added to competent cells. Plates were incubated O/N @ 37°C.
