Penn State iGEM 2014 Notebook Page
Here you will find weekly summaries of our wet laboratory progress, team updates, and accomplishments outside the laboratory. Below [link this] is our detailed, day-to-day progress laboratory notebook.
Weekly Summaries
will create links to each week, but will just list down the page Week 1, Week 2, etc etc
Laboratory Notebook
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Biodetoxification |
Codon Optimization |
| Wednesday, May 21, 2014 |
Emily's first experience with cloning! Ashlee led Emily through several practice experiments from designs made earlier in the year: making a gel, loading samples, gel purifying DNA. |
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| Thursday, May 22, 2014 |
Ashlee and Emily performed a transformation. |
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| Friday, May 23, 2014 |
We picked several colonies for overnight growth to do more cloning tomorrow. |
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| Saturday, May 24, 2014 |
Memorial Day Weekend? How about lab cloning weekend! Ashlee conducted plasmid preparation and digestion. |
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| Monday, May 26, 2014 |
Due to a string of failed clonings with the broadhost vector pSEVA251 and the large inserts, new designs are evaluated! Instead of creating a plasmid with the HMF pathway (7.5 kb) and dCas9 system (5.5 kb), we shall add the HMF pathway and dCas9 to the P. putida genome using homologous recombination. |
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| Tuesday, May 27, 2014 |
Inoculated LB broth with ampicillin and dCas9 plasmid from cryogenic storage; inoculated Lb broth with chloramphenicol and FTV vector from Ashlee's past experiment; streaked the HMF vector on a kanamycin plate. |
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| Wednesday, May 28, 2014 |
Emily and Ashlee made cryogenic storage of the dCas9 plasmid; plasmid prepared the FTV and dCas9 vectors; digested FTV vector; inoculated LB broth with a colony from the HMF plate for overnight growth and plasmid preparation tomorrow. |
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| Thursday, May 29, 2014 |
Prepared plasmid containing the HMF pathway; inoculated LB broth with Lambda Red Recombinase plasmid from cryogenic storage. Ashlee, Emily, and graduate student Iman Farasat ordered primers for three plasmids that will be constructed via Gibson Chew-Back and Annealing Assembly, two of which will be inserted into the genome by homologous recombination. |
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| Friday, May 30, 2014 |
Ashlee and Emily made cryogenic storage of the Lambda Red Recombinase plasmid. |
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| Monday, June 2, 2014 |
Constructed dCas9 gene cassette and plasmid backbone with replication origin ColE1 via PCR Rescue. Gel purified dCas9 and ColE1 cassettes. |
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| Tuesday, June 3, 2014 |
Conducted Colony PCR using P. putida KT2440 strain as DNA template to construct two ~1 kb genome overlaps. Plasmid prepared the Lambda Red Recombinase plasmid, DH10B-PKD46, FTV-ptac-LacI-CmR plasmid, and NoHP_15A_Plmra_CmR plasmid containing RFP with a strong, unique promoter. Stock of NoHP_15A_Pkmra_CmR and FTV_ptac_LacI_CmR for cryogenic storage was also made. Lambda Red Recombinase cassette was amplified using PCR Rescue and gel purified. |
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| Wednesday, June 4, 2014 |
We made ampicillin agar plates and ampicillin antibiotic stock for cloning. The PCR Rescue of Lambda Red Recombinase was also gel purified. |
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| Thursday, June 5, 2014 |
We conducted PCR Rescue to amplify the kanamycin resistance cassette (specifically the neomycin cassette, which also confers resistance to kanamycin) from pSEVA251 KanR plasmid. Two different sets of primers for kanamycin were tested, and the first set was successful - all 4 PCR's were correct. The second set of primers all failed. However, Emily had her first PCR success! |
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| Friday, June 6, 2014 |
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| Saturday, June 7, 2014 |
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| Sunday, June 8, 2014 |
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| Monday, June 9, 2014 |
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| Tuesday, June 10, 2014 |
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| Wednesday, June 11, 2014 |
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| Thursday, June 12, 2014 |
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| Friday, June 13, 2014 |
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| Saturday, June 14, 2014 |
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| Sunday June 15, 2014 |
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| Monday, June 16, 2014 |
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| Tuesday, June 17, 2014 |
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| Wednesday, June 18, 2014 |
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| Thursday, June 19, 2014 |
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| Friday, June 20, 2014 |
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| Saturday, June 21, 2014 |
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| Sunday, June 22, 2014 |
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| Monday, June 23, 2014 |
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| Tuesday, June 24, 2014 |
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| Wednesday, June 25, 2014 |
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| Thursday, June 26, 2014 |
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| Friday, June 27, 2014 |
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| Saturday, June 28, 2014 |
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| Sunday, June 29, 2014 |
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| Monday, June 30, 2014 |
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| Tuesday, July 1, 2014 |
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| Wednesday, July 2, 2014 |
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| Thursday, July 3, 2014 |
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| Friday, July 4, 2014 |
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| Saturday, July 5, 2014 |
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| Sunday, July 6, 2014 |
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| Monday, July 7, 2014 |
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| Tuesday, July 8, 2014 |
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| Wednesday, July 9, 2014 |
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| Thursday, July 10, 2014 |
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| Friday, July 11, 2014 |
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| Saturday, July 12, 2014 |
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| Sunday, July 13, 2014 |
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| Monday, July 14, 2014 |
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| Tuesday, July 15, 2014 |
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| Wednesday, July 16, 2014 |
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| Thursday, July 17, 2014 |
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| Friday, July 18, 2014 |
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| Saturday, July 19, 2014 |
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| Sunday, July 20, 2014 |
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| Monday, July 21, 2014 |
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| Tuesday, July 22, 2014 |
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| Wednesday, July 23, 2014 |
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| Thursday, July 24, 2014 |
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| Friday, July 25, 2014 |
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| Saturday, July 26, 2014 |
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| Sunday, July 27, 2014 |
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| Monday, July 28, 2014 |
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| Tuesday, July 29, 2014 |
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| Wednesday, July 30, 2014 |
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| Thursday, July 31, 2014 |
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