Team:Tsinghua-D/Notebook.html

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        <a href="#21"><strong>Literature research</strong> : Feburary - April<a>      </div>
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      <div id="Layer12"><a href="#41"><strong>Dry Experiment</strong>: April - August<a> </div>
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             <p align="left" class="STYLE2 STYLE4"><span class="STYLE2">2012.2-2012.4<br>
             <p align="left" class="STYLE2 STYLE4"><span class="STYLE2">2012.2-2012.4<br>
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             Conduct literature research.</span></p>
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             Conduct literature research.</span><br>
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             <p align="left" class="STYLE2 STYLE4"><span class="main">2012.4-2012.8<br>
             <p align="left" class="STYLE2 STYLE4"><span class="main">2012.4-2012.8<br>
             Learn Vienna RNA Package.<br>
             Learn Vienna RNA Package.<br>
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Latest revision as of 22:37, 26 September 2012



The Gantt Chart of the project



Experimental log

 

 

 

 

2 August
Sun Mon Tue Wed Tue Fri Sat
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31  
September 1
Sun Mon Tue Wed Tue Fri Sat
30     1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29

2012.2-2012.4
Conduct literature research.

2012.4-2012.8
Learn Vienna RNA Package.
Learn physical and biological background knowledge.
Write RNAThermo.

2012.8.14

Get ‘1st RNAT + eGFP’ gene from the standard part BBa_I714891 by a three-round overlapping PCR. The result of the first round is negative due to insufficient DNA templates.
Prepare for transformation of the standard part BBa_I714891 to bacteria.



2012.8.15

Transform the BBa_I714891 to the bacteria
Pick single colony of the bacteria harboring BBa_I714891 plasmid.


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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