"

From 2012.igem.org

Monday:

• Band purification of LuxCD & Lux EG.

• Gel electrophoresis: Colony PCR of RS2 cut (no extra restriction site) and Luc CDEG. Right sized bands: 11 and 12. Plasmids from preceding colonies were extracted and sized was checked by electrophoresis. Finally they were ligated to B0014 and B0015.

Tuesday:

• PCR Run

VB psb1C3, VBpsb1A3, VB psb1T3, VB psb1K3, V psb1K5, Lux CD

• Colony PCR

RS1+RS2: (12 colonies selected) B0014+RS2: (12 colonies selected) B0015+RS2: (12 colonies selected) Lux CD+Lux EG: (4 colonies selected) Positive and negative control

Results: correct controls. Most of the colonies were right sized. Error in Lux CD+EG because the bands were much smaller than expected (1000 bp). Two colonies were chosen in each case. Digestions left overnight: Lux CD from Brick, and Lux EG obtained by PCR. Wrong digestions were eliminated.

Wednesday:

Overnight digestions were finished and 20 μL were run in gel (1%). Digestions showed right size and high DNA concentration. Part Lux CDEG was ligated, transformed and left growing.

• Colony PCR purification of slected colonies

RS1+RS2 (colonies 2 and 6) B0014+RS2 (colonies 2 and 6) B0015+RS2 (colonies 20 and 23)

Desired parts were obtained.

• Digestions

RS1+RS2 (E+P) B0014+RS2 (X+P) B0015+RS2 (X+P)

With the last two and RS1+Kan (E+s). The following parts were ligated and transformed:

RS1+Kan+B0014+RS2 in pSB1C3 RS1+Kan+B0015+RS2 in pSB1C3

These were used to insert parts between RS1 and Kan through Gibson Assembly.

• Amplication and purification of plasmids: PSB1C3, pSB1T3, pSB1K3, pSB1A3, pSB1K. They were very faint and their DNA concentration was vwery low, therefore the plasmids were dismissed. The only one that was kept was pSB1K5 (24ng/μl)

• PCR run:

Name PCR Template Primer F Primer R

B1C VB-pBAD LuxBrick 16.F7 16.F10 B2C VB-pBAD LuxBrick 16.F7 16.G2

B1	ADF3	          ADF3	                16.F9	        16.F8	   
B2	ADF3	          ADF3	                16.G1	        16.F8	   

C1 RS1+RS2 RS1+RS2 14.F2 14.G2

C1A	pSB1A3 for gibson Linearized VB pSB1A3	14.F3	        14.G1	   
C1T	pSB1T3 for gibson Linearized VB pSB1T3	14.F3	        14.G1	   

LCD Lux CD Lux CD VF2 VR pSB1A3 pSB1A3 Linearized VB pSB1A3 Suffix F Preffix R

Notes B1C, B2C, C1, LCD and pSB1A3 were right sized and were miprepped afterwards (elution in 20 μL). Only B1C (VB-pBAD) and RS1+RS2 yielded high concentrations (25,9 ng/μL and 73,1 ng/μL) respectivamente. A new PCR will be done to amplify mipreps with low concentrations.