Lab Aug 20 2009

9.20 am

Assembly Plates

- We have colonies on every plate! They're all tiny right now but they were plated only 14 hours ago!

- Pick colonies from any assembly plates that grew overnight and broth culture overnight for miniprep on Friday

Checking on broth cultures

- check if the overnight cultures of K098988 and K081005 grew ->miniprep and gel

Gel

- Ran four of the five digests (R0010, J23066, J23102, E0422, no room for P1010 Cm) and the 3 ligation mixes (A, B, C)

- A = K235000 = R0010+J23066 --> pLac+ribokey

- B = K235001 = J23102+J23066 --> constitutive promoter+ribokey

- C = K235002 = J23102+E0422 --> constitutive promoter+ECFP

Started at 10.10 am - Finished at 10.55 am 1 Kb ladder 	A 	B 	C

U

J23102

U

R0010

D

E0422

D

J23066 Faint Band 	Faint Band 	Faint Band 	Faint Band 	Faint Band 	Faint Band 	Band

iGEM gel 4.tif

Gels that have not been run:

- K145303 Quad (was miniprepped but not enough room on the gel yesterday)

- P1010 Cm Plasmid digest (not enough room today)

- K098988 and K081005 (miniprepped today)

- P1010 Amp (was miniprepped but not run)

- R0010 (pLac), J23066(ribokey + terminator), C0051 (λcI repressor), I13521 (Ptet RFP generator) (was miniprepped but not run)

Possible assemblies to do

-C0051 (λcI CDS)+B0015 (double transcription stop)

- J06504+B0015 --> mCherry+stop,

- R0010+B0034 --> pLac+RBS

- J23102 (const. promoter)+ J23032 (ribolock)

P0151 (PoPs cI lambda)

Assemblies Today New Part Number Upstream 	Downstream Destination Plasmid E + S 	X + P 	E + P K235003 	C0051 A 	B0015 AK 	P1010 C K235004 	R0010 A 	B0034 A 	P1010 C K235005 	J6504 A 	B0015 AK 	P1010 C


 * So for some reason we only did three, while we could have done the fourth one as well.

Started 3A Assembly Protocol

Iinital digest sat in the 37 far longer than the times in the prootcol. Closer to 40 min instead of 15....

2nd incubation started in about 75 degree water.

We let the epis sit for 5 min before continuing with the ligation.

Room temp incubation at 2:20 pm

80oC incubation at 2:31pm

Started Transformation Protocol at 3.00 pm

Note to selves: make sure the 80C bath is turned on to heat at LEAST an hour before you start the assembly procedure. Preferably more if it's coming from room temperature.

Borth cultures were started from each of yesterday's tranformations:

- A = K235000 = R0010+J23066 --> pLac+ribokey

- B = K235001 = J23102+J23066 --> constitutive promoter+ribokey

- C = K235002 = J23102+E0422 --> constitutive promoter+ECFP

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