2012.8.16
Conduct the first round of overlapping PCR of the gene ‘1st RNAT + eGFP’.
Culture the bacteria harboring BBa_I714891 plasmid.
2012.8.17
Extract BBa_I714891 plasmid from bacteria.
Digest the obtained BBa_I714891 plasmid with Pst1 and EcoR1, however the band is unclear.
Take the BBa_I714891 plasmid as template to do the first round overlapping PCR of the gene ‘1st RNAT + eGFP’.
A gradient of PCR annealing temperatures of the gene ‘1st RNAT + eGFP’ are tested to find the optimal one.
2012.8.18
Purify product of the first round overlapping PCR of the gene ‘1st RNAT + eGFP’.
Repeat the PCR and electrophoresis verification procedure.
2012.8.19
Conduct the second round overlapping PCR of the gene ‘1st RNAT + eGFP’.
61℃ is proved to be the best PCR annealing temperature of the gene ‘1st RNAT + eGFP’.
2012.8.20
Conduct the third round overlapping PCR of the gene ‘1st RNAT + eGFP’ and electrophoresis verification procedure.
2012.8.21-8.22
Purify the product of the third round overlapping PCR of the gene ‘1st RNAT + eGFP’ by gel extraction.
2012.8.23
Digest the plasmid and the PCR product of the gene ‘1st RNAT + eGFP’ with EcoR1 and Pst1.
Ligate the two digested DNA.
2012.8.24
Test shows the construction of the plasmid fail.
Retry the ligation
2012.8.25
Ligation and transformation of ‘the 1st RNAT + eGFP’ are done.
Replicate pSB1C3 plasmid with the protocol given by iGEM.
Adapt pET-Duet plasmid as expression vector instead of pSB1C3.
Prepare for the lysozyme experiment.
Transformation of the pSB1C3 plasmid is done.
2012.8.26
Pick the single and positive colony of ‘1st RNAT + eGFP’.
2012.8.27
Get ‘signal peptide + lysozyme’ gene by a three-round overlapping PCR.
Conduct 1st and 2nd round of the overlapping PCR of the gene ‘1st RNAT + signal peptide + lysozyme’.
2012.8.28
Amplify the PCR product of the gene ‘1st RNAT + signal peptide + lysozyme’.
2012.8.29
Prepare for the in-line probing.
2012.8.30
Conduct the in-line probing.
Prepare for the ‘2nd RNAT + eGFP’ and ‘3rd RNAT + eGFP’ experiments.
2012.8.31
Learn fluorescence signal detection and RNA in vitro transcription procedure in the in-line probing method.
Conduct the ‘2nd RNAT + eGFP’ and ‘3rd RNAT + eGFP’ experiments.
2012.9.1
Send the plasmid harboring ‘1st RNAT + eGFP’ gene for sequencing
The RNA in vitro transcription is done.
Conduct the ‘2nd RNAT + eGFP’ and ‘3rd RNAT + eGFP’ experiments.
Conduct the ‘1st RNAT + signal peptide + lysozyme’ experiment.
2012.9.2
Send the plasmid harboring ‘2nd RNAT + eGFP’ gene and ‘3rd RNAT + eGFP’ gene for sequencing.
Conduct the ‘1st RNAT + signal peptide + lysozyme’ experiment.
2012.9.3
Overlapping PCR of the ‘1st RNAT + signal peptide + lysozyme’ gene is done.
Obtain ‘1st RNAT + eGFP’ result, however not convincing.
2012.9.5
Obtain ‘2nd RNAT + eGFP’ result, convincing result.
Obtain ‘3rd RNAT + eGFP’ result, convincing result.
2012.9.8-9.22
Repeat ‘1st RNAT + eGFP’, ‘2nd RNAT + eGFP’ and ‘3rd RNAT + eGFP’ results.
Conduct the in-line probing.
Standard parts are made and sequenced.
2012.9.23-9.26
Process the data, write for the report.
Prepare for the ‘2nd RNAT + signal peptide + lysozyme’ and ‘3rd RNAT + signal peptide + lysozyme’ experiments.
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