IGEM:Virginia/2012/Notebook/Genetically engineered bacteriophage for diagnosis of whooping cough/2012/09/23

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Week of 9/23/12

Redid backbone (with both E+S and E+P) but CIAPing the vector prior to ligation in order to decrease the chance of the empty vector ligating to itself and creating false positive colonies, producing acceptable concentrations of 90.4 and 71.0 ng/μL, respectively. Colonies for the bbp9, hCG, SynCG+promotor, SynCG+promotor+RBS,, plasmid (E+S), and plasmid (E+P) all are acceptable in the double digits. The SynCG colony is still in the thousands. Inoculated colonies, performed plasmid prep, and used PCR to confirm presence of inserts. hCG, SynCG, and P+RBS+SynCG all have exceptionally large yields over 5000 ng/μL.
EtBr gel confirms presence of hCG biobrick and SynCG biobrick.
Extracted SynCG (E+X) with phenolchloroform, resulting in similarly high yields.
Attempted ligations of bbp9 biobrick but only empty vectors were produced.
Transformed DH5α with ligations and plated on LB with chlor and made inoculations of bbp9 colonies and incubated at 37°C. The colonies later grew and were turned into 5 5mL cultures.
Plasmid prep for 5 bbp9 inoculations and 5 P+RBS+SynCG inoculation and then run PCR and gel. All colonies were acceptable with a concentration of at least 3000 ng/μL.
Gel indicates that the fourth bbp9 PCR may have a possible biobrick as well as the first P+RBS+SynCG.
Restarted with 6 tube of T7 digest with BclI, followed by ethanol precipitation cleanup and a PCR cleanup kit. Cleaned up the T7 halves and resuspended them in 20 μL of EB buffer, resulting in a final concentration of 623,1 ng/μL.
Performed PCR amplification of promotor, RBS, SynCG after plasmid prep of DH5α transformed with biobrick plasmid. No evidence of primers appeared after running the PCR products on a 2% agarose gel, suggesting that the PCR mixture was incorrect.
Performed 12 inoculations of P+RBS+SynCG colonies. After running these colonies through a PCR, the nanodropping of these plasmid preps confirmed that all 12 had acceptable concentrations in the upper double digits.