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| | |align="center" width="150pt"|{{#calendar: title=Virginia_Commonwealth |year=2009 | month=10}} | | |align="center" width="150pt"|{{#calendar: title=Virginia_Commonwealth |year=2009 | month=10}} |
| | |} | | |} |
| - | ==Thursday 23 July 2009== | + | ==Friday 24 July 2009== |
| | ===Results=== | | ===Results=== |
| - | ''Maria and Afton'' | + | ''Craig and Clay'' |
| - | * All overnight cultures grew except one vial of NEB 10 beta seed stock | + | * A single colony was found growing on the plate containing cells transformed with part BBa_I742111. All other plates showed no growth. |
| - | ** there was a second vial of seed stock that did grow successfully
| + | |
| - | * Transformations were successful
| + | |
| - | {| cellpadding="10" align="center" border="0.9" padding="5"
| + | |
| - | |+
| + | |
| - | |-
| + | |
| - | ! 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 ||
| + | |
| - | |}
| + | |
| - | [[User:Trentay|Trentay]] 17:19, 23 July 2009 (UTC)
| + | |
| | ---- | | ---- |
| | + | ''Kevin & Adam'' |
| | + | *PCR went well. |
| | + | *Gel showed correct results |
| | + | *purification went well, amount of DNA collected will be found Monday before digestion. |
| | + | -[[User:Bussingkm|Bussingkm]] 22:03, 24 July 2009 (UTC) |
| | + | |
| | ===Tasks=== | | ===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
| |
| - | [[User:Trentay|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. [[User:Bussingkm|Bussingkm]] 20:56, 23 July 2009 (UTC)
| |
| - |
| |
| | ''Craig and Clay'' | | ''Craig and Clay'' |
| - | * Electrotransformation of the limonene pathway parts will be attempted a third time. | + | * The parts displaying no growth will be left over the weekend to grow. The plate with part BBa_I742111 will be left in the refrigerator until Monday, at which time a miniprep will be performed. |
| | ---- | | ---- |
| | ====Wetlab==== | | ====Wetlab==== |
| - | ''Maria and Afton''
| + | ''Kevin & Adam'' |
| - | * Cryogenic stocks were made
| + | *PCR padding regions onto promoters |
| - | * Overnight culture was made of J23100 w/ J06702 RET in LB+Cm
| + | *purify PCR products |
| - | [[User:Trentay|Trentay]] 21:25, 23 July 2009 (UTC)
| + | *run gel to test PCR products |
| - | | + | |
| - | ''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.
| + | |
| - | | + | |
| - | {| cellpadding="10" border="1" alignment="center"
| + | |
| - | |##|| ~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.
| + | |
| - | | + | |
| - | -[[User:Bussingkm|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.
| + | |
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