Team:Penn State/Notebook

From 2012.igem.org

(Difference between revisions)
Line 266: Line 266:
<!--- Sgt_Bisendi -->
<!--- Sgt_Bisendi -->
</p>
</p>
 +
<p>
 +
<b>Thursday, June 14, 2012</b><br/>
 +
Plasmids from Hannah's CBAR reaction were extracted and back-up cultures were inoculated. Victoria, Kait, and Alexander ran a gel to confirm the components of plasmids to be used in the final Codon Optimization Construct. Kevin and Chris inoculated a culture with a colony containing the vector plasmid for the Multidirectional Promoters Project. They came in later that night to extract the plasmid.
 +
<!-- Sgt_Bisendi -->
 +
</p>
 +
<p>
 +
<b>Friday, June 15, 2012</b><br/>
 +
Kevin ran a PCR on the vector component that he and Chris extracted the previous night. This was run through gel electrophoresis to separate the desired component from other side-products, and to ensure the fragment was the correct size. This was finally cleaned and concentrated for a CBAR reaction on Monday. Hannah grew her cultures from her CBAR reaction to get a higher plasmid yield. She also prepared new chemically competent DH10B cells since the stock was running low. Victoria, Kait, and Alexander checked a plasmid's components for the Codon Optimization Project.
 +
<!-- Sgt_Bisendi -->
 +
</p>
 +

Revision as of 20:08, 15 June 2012

Penn State iGEM 2012


Penn State Notebook

Week 1

Monday, May 22, 2012
We prepared ampicillin and streptomycin plates in preparation for production of plasmids and competent cells.

Tuesday, May 23, 2012
Inoculation of LB broth with DH10B E. coli strain to prepare master mix/culture of cells in preparation for making chemically competent cells. These cells were incubated throughout the day and prepared for competency by the end of the day. Promotes were also planned out for the Multiple Promoters Project and ordered. These promoters were designed to combine some of the components of the plasmid.

Wednesday, May 24, 2012
Biobricks were hydrated and transformed into Chemically Competent cells through heat shock ( BBa_R0063, BBa_R1062, BBa_R0078, BBa_I1051, BBa_R0079, BBa_R0071 ). LOB broth was also prepared for the recovery from the transformation. These cells were then plated to produce isolated colonies.

Thursday, May 25, 2012
Additional Biobricks were hydrated and transformed into Chemically Competent cells through heat shock ( BBa_I712074, BBa_R0062, BBa_I746104, BBa_I719005, BBa_J23119, BBa_J23100, BBa_J23101, BBa_J23102, BBa_J23103, BBa_J23104, BBa_J23105, BBa_J23106, BBa_J23107, BBa_J23110 ). LOB broth was used to help the cells recover from their transformation. More agar plates were poured. The Transformed cells form yesterday yielded isolated colonies. A single colony from each plate was used to create a broth in preparation for mini-prep of the plasmids.

Friday, May 25, 2012
Miniprep was done to extract the first six plasmids we transformed ( BBa_R0063, BBa_R1062, BBa_R0078, BBa_I1051, BBa_R0079, BBa_R0071 ). Colonies that grew from the transformation of our last 14 plasmids ( BBa_I712074, BBa_R0062, BBa_I746104, BBa_I719005, BBa_J23119, BBa_J23100, BBa_J23101, BBa_J23102, BBa_J23103, BBa_J23104, BBa_J23105, BBa_J23106, BBa_J23107, BBa_J23110 ) were cultured for later plasmid extraction.

Saturday, May 26, 2012
Hannah and Kevin extracted the plasmids from the 14 colonies that were cultured yesterday. Two of the BioBricks that needed to be retransformed ( BBa_J23102, BBa_J23105) had isolated colonies on their plates and were cultured by Kevin for plasmid extraction the next day.


Week 2

Sunday, May 27, 2012
Hannah and Kevin extracted the two cultures that Kevin prepared yesterday. These plasmids were put on ice for concentration analysis later with the other extracted plasmids.

Monday, May 28, 2012
Memorial Day

Tuesday, May 29, 2012
The 20 plasmids that have been grow during the previous week and weekend were digested by Hannah, Victoria, and Kevin. The plasmids were run in a gel, however errors in the gel's preparation resulted in a poor run. Always make sure your gasket is correctly seated before you pour the agarose!

Wednesday, May 30, 2012
Plasmids for the Multidirectional Promoters Project were transformed and cultured by Hannah and Kevin. Chris, Kait, and Victoria re-transformed 10 plasmids and cultured those. A gel was run to confirm our plasmids and fragments but was inconclusive due to low concentration.

Thursday, May 31, 2012
Fragments for the Multidirectional Promoter Project were digested and run through PCR by Hannah and Kevin. Kait and Victoria re-transformed the remaining 10 plasmids and 1 from yesterday that did not yield colonies. Kait and Victoria ran a gel to confirm a plasmid they could use in the Codon Optimization Project.
The originally transformed promoters were disposed of. Some of the cultures became a purple color. After centrifuging it was shown that the bacteria themselves were purple. This may be from contamination, so to be on the safe side, these plasmids were re-transformed. This may be an artifact from the plasmids themselves, but there is no consistent pattern. Very strange indeed! Always remember to use good sterile techniques when working with media and cultures!

Friday, June 1, 2012
Today Hannah digested components for the Multiple Start Codon Project. Later she ran a gel and extracted the components for later use. Kait and Victoria prepared a plasmid for the Codon Optimization Project. Kevin and Chris began work on a plasmid backbone and fluorescent component of the Multidirectional Promoters Project.
The team also received a new gel illuminator to see their bands without the use of UV light. They were very excited.


Week 3

Monday, June 4, 2012
Hannah hydrated BBa_I13521 and used it to gauge the competency of the chemically competent cells we prepared earlier. Victoria and Kait ran a gel to confirm some of the components they need for the Codon Optimization Project. Kevin and Chris worked on some cleaning and housekeeping.

Tuesday, June 5, 2012
Victoria and Kait completed a transformation for the Codon Optimization Project. Kevin mini-prepped two plasmids for the Multidirectional Promoters Project. Chris and Kevin then froze some cultures containing these plasmids for later use. Hannah completed a CBAR and transformation for the Multiple Start Codon Project. She also tested the competency rate of the chemically competent cells the team prepared earlier.

Wednesday, June 6, 2012
Kevin and Chris ran a gel to separate components for the Multidirectional Promoters Project's final construct. Victoria and Kait inoculated cultures with isolated colonies. This culture has a plasmid that they will use for their final construct. Hannah is in the process of building a higher efficiency Chemically Competent DH10B cell line. The higher transformation rate will help not only the team but is critical for her CBAR assembly of her construct for the Multiple Start Codon Project. Hannah also started planning and development of a secondary construct for the Multidirectional Promoters Project to act as a control for the experiments.

Thursday, June 7, 2012
Kevin and Chris ran a gel to separate another component for the Multidirectional Promoters Project. One of the component did not run well on the gel. This component was amplified again with PCR. Kait and Victoria continued work on the Codon Optimization Project with the digestion of their plasmid, and subsequent gel extraction of the specific components. Hannah continued work on designing the primers for the CBAR reaction to create the control construct for the Multidirectional Promoters Project. Hannah also prepped a culture in an attempt to get a stock culture of DH10B growing. Her goal is to create a higher efficiency Chemically Competent (CC) DH10B cell line to help her complete her CBAR for her Multiple Start Codon Project.

Friday, June 8, 2012
Kevin extracted components for the Multidirectional Promoters Project. from the gel that was run yesterday. Hannah worked more on the design of the primers for one of the constructs for the Multidirectional Promoters Project. Kevin completed a streak plate to create isolated colonies in preparation for the production of more Chemically Competent cells. Kait and Victoria continued their work on their construct for the Codon Optimization Project. Victoria designed primers for their construct.

Saturday, June 9, 2012
Kevin and Chris produced a new set of Chemically Competent cells. Their goal is to create a cell line with a higher transformation efficiency.


Week 4

Monday, June 11, 2012
Victoria and Kait mini-prepped a plasmid for their final construct. Hannah and Victoria poured new chloramphenicol plates. Chris ran a digest on six plasmids for the Multidirectional Promoters Project. Kevin and Hannah transformed six plasmids for this project as well. Kevin ran a transformation to test the competency of the cells that he and Chris prepared over the weekend.

Tuesday, June 12, 2012
Hannah completed her CBAR transformation today. Kait, Victoria, and Alexander completed a gel, gel extraction, and transformation for constructs for the Codon Optimization Project. Kevin and Chris inoculated six cultures with transformed colonies that grew overnight. Chris also did a comparison transformation between the CC DH10B cell line that he and Kevin made over the weekend, and the other CC cells that the team has been using.

Wednesday, June 13, 2012
Victoria and Kait made more plasmid stock for the Codon Optimization Project. Hannah made cultures from the colonies that grew from her CBAR reaction. Kevin and Chris extracted plasmids for the Multidirectional Promoters Project. They also began making more plasmid vector in preparation for their CBAR reaction.

Thursday, June 14, 2012
Plasmids from Hannah's CBAR reaction were extracted and back-up cultures were inoculated. Victoria, Kait, and Alexander ran a gel to confirm the components of plasmids to be used in the final Codon Optimization Construct. Kevin and Chris inoculated a culture with a colony containing the vector plasmid for the Multidirectional Promoters Project. They came in later that night to extract the plasmid.

Friday, June 15, 2012
Kevin ran a PCR on the vector component that he and Chris extracted the previous night. This was run through gel electrophoresis to separate the desired component from other side-products, and to ensure the fragment was the correct size. This was finally cleaned and concentrated for a CBAR reaction on Monday. Hannah grew her cultures from her CBAR reaction to get a higher plasmid yield. She also prepared new chemically competent DH10B cells since the stock was running low. Victoria, Kait, and Alexander checked a plasmid's components for the Codon Optimization Project.