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Virginia Commonwealth/23 July 2009
From 2009.igem.org
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''Kevin and Adam'' | ''Kevin and Adam'' | ||
*We need to PCR DNA padding onto the first nine synthesized promoters. When we ordered these oligo's we did not consider that we needed additional overhang from the BioBrick prefix and suffix in order for the restriction enzymes to work properly. Doing this PCR will correct that error. [[User:Bussingkm|Bussingkm]] 20:56, 23 July 2009 (UTC) | *We need to PCR DNA padding onto the first nine synthesized promoters. When we ordered these oligo's we did not consider that we needed additional overhang from the BioBrick prefix and suffix in order for the restriction enzymes to work properly. Doing this PCR will correct that error. [[User:Bussingkm|Bussingkm]] 20:56, 23 July 2009 (UTC) | ||
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| + | ''Craig and Clay'' | ||
| + | * Electrotransformation of the limonene pathway parts will be attempted a third time. | ||
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====Wetlab==== | ====Wetlab==== | ||
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-[[User:Bussingkm|Bussingkm]] 21:04, 23 July 2009 (UTC) | -[[User:Bussingkm|Bussingkm]] 21:04, 23 July 2009 (UTC) | ||
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| + | ''Craig and Clay'' | ||
| + | * Electrotransformation of the limonene parts was reattempted. Fifty microliters were streaked on each ampicillin-selective plate. | ||
Revision as of 14:23, 24 July 2009
Contents |
Thursday 23 July 2009
Results
Maria and Afton
- All overnight cultures grew except one vial of NEB 10 beta seed stock
- there was a second vial of seed stock that did grow successfully
- Transformations were successful
| Name | Type | Plate | Purpose | Growth Observation | |
|---|---|---|---|---|---|
| NEB 10 beta | seed stock | LB | make more seed stock | lawn | |
| NEB 10 beta | electro comp. | LB | (+) Control/ make more stock | lawn | |
| NEB 10 beta | electro comp. | LB | (+) Control shocked | lawn | |
| DB 3.1 | electro comp. | LB | (+) Control shocked | lawn | |
| DB 3.1 | electro comp. | LB | (+) Control/make more stock | lawn | |
| NEB 10 beta | electro comp. | Cm | (-) Control | no growth | |
| NEB 10 beta | electro comp. | Cm | J23100 w/ J06702 | 6 small colonies |
Trentay 17:19, 23 July 2009 (UTC)
Tasks
Maria and Afton
- Make cryogenic stocks of all overnight cultures
- Make stocks of parts I1352 (RFP) and I13522 (GFP)
- Update notebook, documents
- Pick colonies from transformation
- pick controls as well to make more seed and electrocompotent stock
Trentay 17:25, 23 July 2009 (UTC) Kevin and Adam
- We need to PCR DNA padding onto the first nine synthesized promoters. When we ordered these oligo's we did not consider that we needed additional overhang from the BioBrick prefix and suffix in order for the restriction enzymes to work properly. Doing this PCR will correct that error. Bussingkm 20:56, 23 July 2009 (UTC)
Craig and Clay
- Electrotransformation of the limonene pathway parts will be attempted a third time.
Wetlab
Maria and Afton
- Cryogenic stocks were made
- Overnight culture was made of J23100 w/ J06702 RET in LB+Cm
Trentay 21:25, 23 July 2009 (UTC)
Kevin and Adam
- First we reconstitute the DNA. The rule of thumb is that you take 1/2 the amount of oligo's (nmol) of H2O in uL for ~100x DNA stock solution. Store in (-20C)
- A problem was encountered while reconstituting the DNA. For several of the sequences the amount of water described above was not sufficient to reconstitute the DNA. It was too viscus and could not be pipetted at all. We doubled the amount of water that was used and made note of the approximate concentration.
| ## | ~nmol DNA | uL H2O | Concentration |
| Design 1 | 25 nmol | 12.5 uL | 100x |
| Design 2 | 25 nmol | 25 uL | 50x |
| Design 3 | 25 nmol | 25 uL | 50x |
| Design 4 | 25 nmol | 12.5 uL | 100x |
| Design 5 | 25 nmol | 25 uL | 50x |
| Design 6 | 25 nmol | 25 uL | 50x |
| Design 7 | 25 nmol | 25 uL | 50x |
| Design 8 | 25 nmol | 25 uL | 50x |
| Design 9 | 25 nmol | 25 uL | 50x |
| Forward end padding | 48.3 nmol | 24.15 uL | 100x |
| Reverse end padding | 36.4 nmol | 18.2 uL | 100x |
- This DNA was frozen in Adam & Kevin's stock box in the vials that the DNA was shipped in.
- Each sample of DNA was diluted to 1x and 100 uL with the appropriate amount of nanopure H2O to be used for PCR.
- The first round of PCR was done using the standard protocol. There was an error when the machine was started. It somehow was paused while the temperature was 95C. This was discovered long after it occurred. Deng advised us to start over. We both have prior commitments and thus cannot stay late enough to start over. We will start over early in the morning. dNTP is made and mixed in a 1:1:1:1 ratio.
-Bussingkm 21:04, 23 July 2009 (UTC)
Craig and Clay
- Electrotransformation of the limonene parts was reattempted. Fifty microliters were streaked on each ampicillin-selective plate.
