Week14 9/12/10-9/18/10

From 2010.igem.org

Week14 Highlights

We ran multiple gels to test our LacP-RBS and LacP-RBS-CHS3 assemblies. We continued preparing and assembling our combinations of Cp-LacPi-GFP for characterization. We also poured more Chlor and Tet plates using smaller (60mm x 15mm) petri dishes given to us by VWR.

9/13/10

Assembled LacP-RBS-CHS3 using standard assembly. Grew on Chlor plates.

9/14/10

Inoculated Apop1

Inoculated 18 colonies of LacP-RBS-CHS3 for screening purposes

Plated CHS3,CP-LacPI-GFP, CP-LacPI-RFP.

9/15/10

Miniprepped LacP-RBS-CHS3 and Apop1

Digested LacP-RBS-CHS3 with X and P (CHS3 contains an internal P site)

Kevin put 15 overnight cultures into the 37 degree incubator at 6pm.

Kevin the CP-LACPI assemblies as well as the CP-LACPI-GFP/RFP assemblies around 9 pm. There was one normal size KAN plate for each CP-LACPI-GFP/RFP assembly (including a negative control) and two small size TET plates for each CP-LACPI assembly (with only one negative control plate).

Timi poured two sleeves of mini-Chlor and one sleeve of mini-Tet plates.

9/16/10

Ran digested LacP-RBS-CHS3 on a gel:

Majority contained 1 band around 3600bp
3 contained 3 bands (2000bp, 1600bp, 1100bp)

Timi mini-prepped the 15 overnight cultures that Kevin put into the incubator last night.

9/17/10

Digested LacP-RBS(1), LacP-RBS(2), and LacP-RBS(3) with ES --> Ran on a gel LacP-RBS(2) and LacP-RBS(3) showed correct bands at ~70bp

Timi miniprepped the cultures Kevin inoculated last night. Note: the tube caps were snapped shut while they were shaking-- that probably affected the DNA concentration of some of the minipreps.

Results: Two of the cultures did not have any cells so he probably just missed the colony or something- not a big deal.

CP3-LacPi2 retrans:

1) 155.3

2) 135.1

3) 76.8

CP1-LacPi2 seq:

1) 102.9

2) 91.7

3) 71.7

CP2-LacPi2:

1) 83.8

2) 89.6

CP1-LacPi2

1) 54.7

2) 94.7

CP1-LacPi1:

1) 108.8

2) 66.0

3) 36.5

The concentrations are all on the lower side compared to what we have been getting. I would say to just send some out for sequencing to see if the ligations actually worked before worrying about the concentrations. These DNA vials are in my DNA box in the -20.

PLATES:: The CP-LACPI assemblies as well as the CP-LACPI-GFP/RFP assembly plates still had really small colonies on them. Timi came back later to put them into the cold room.

Kevin put in another series of CP-LacPI-GFP/RFP incubations at around 6pm.