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
Comments (2)
Kyliah Clarkson said
at 4:10 pm on Aug 20, 2009
Ky says sorry to Derek for having her cell phone off :S I was working on my powerpoint thingy in the library this morning and forgot to turn my cell back on.
james jacoby said
at 6:09 pm on Aug 20, 2009
Sorry Ky, I'm not one to talk about cell phone availability! I think we're meeting again at around 9, so come on in whenever you like.
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