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
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
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.