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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.