Difference between revisions of "Team:NCKU Tainan/Notebook Construction"

 
Line 85: Line 85:
 
                         <div class="title-line long" id="sec5"></div>
 
                         <div class="title-line long" id="sec5"></div>
 
                         <div class="title-content">We transformed BioBricks from the 2016 igem distribution<br>
 
                         <div class="title-content">We transformed BioBricks from the 2016 igem distribution<br>
               (BBa_J23110, BBa_J23101,BBa_B0031,BBa_B0032,BBa_E1010)</div>
+
               (BBa_J23110, BBa_J23101, BBa_B0031, BBa_B0032, BBa_E1010)</div>
 
                         <div class="text-content">
 
                         <div class="text-content">
 
                           <h5>Jun. 03/2016</h5>
 
                           <h5>Jun. 03/2016</h5>

Latest revision as of 16:10, 19 October 2016

Notebook Construction - iGEM NCKU

NOTE / Construction
Notebook - Construction
Extracted plasmid from the bacteria provided by XMU
(pSB1C3-BBa_B0034, pSB1C3-BBa_B0015, pSB1C3-BBa_K592009)
Mar. 30/2016

We got the bacteria with Biobrick from XMU.

Apr. 01/2016

Pre-culture: 3 ml of LB (with Chloramphenicol) was inoculated with one colony from LB agar plate.

Apr. 02/2016

Culture: 10 ml of LB (with Chloramphenicol) culture with 100 μL pre-cultured bacteria.

And grow up at 37 degree for 10-12 hours.

Apr. 03/2016

Extracted the plasmid, and measured the concentration.

Store the plasmid at -20 degree.

Got the glucose sensitive promoter (PI, Pcar) by PCR
Apr. 16/2016

PCR for PI & Pcar.
Result: After gel purified, the concentration of the DNA was too low to do construction.

Apr. 17/2016

PCR for PI & Pcar again (in big scale).

Purified The PCR product and cloned to T-vector.

Apr. 18/2016

Confirmed the white colony by PCR (using M13F & M13R as the primer)
Result: Some of the colonies were correct.

Pre-culture: 3 ml of LB (with Ampicillin) was inoculated with one correct colony from the agar plate.

Apr. 19/2016

Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pMD19T-PI and pMD19T-Pcar were correct.

Apr. 20/2016

Culture: 10 ml of LB (with Ampicillin) cultured with 100 μL pre-cultured bacteria.

Grow up at 37 degree for 8 hours and stored the bacteria at -70 refrigerator.

Constructed pSB1C3-B0034-K592009
Apr. 30/2016

Digested pSB1C3-B0034 (SpeI,PstI) and pSB1C3-K592009 (XbaI,PstI)

Ligated K592009 (insert) to pSB1C3-B0034 (vector) and transformed the product.

May 01/2016

Confirmed by colony PCR (using Prefix-F & Suffix-R as the primer).

Pre-culture: 3 ml of LB (with Chloramphenicol) was inoculated with one correct colony from the agar plate.

May 02/2016

Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pSB1C3-B0034-K592009 was correct.

May 03/2016

Culture: 10 ml of LB (with Chloramphenicol) cultured with 100 μL pre-cultured bacteria.

Grow up at 37 degree for 8 hours and stored the bacteria at -70 refrigerator.

Construct pMD19T-Pcar-B0034-K592009 and pMD19T-PI-B0034-K592009
May 07/2016

Digested pSB1C3-B0034-K592009 (XbaI,PstI), pMD19T-Pcar (SpeI,PstI) and pMD19T-PI (SpeI,PstI)

Ligated B0034-K592009 (insert) to pMD19T-Pcar (vector) and pMD19T-Pcar (vector).

Transformed the product.

May 08/2016

Confirmed by colony PCR (using M13F & M13R as the primer).

Pre-culture: 3 ml of LB (with Ampicillin) was inoculated with one correct colony from the agar plate.

May 09/2016

Extracted plasmid and confirm plasmid size by gel electrophoresis.
Result: The size of pMD19T-Pcar-B0034-K592009 and pMD19T-PI-B0034-K592009 were correct.

May 10/2016

Culture: 10 ml of LB (with Ampicillin) cultured with 100 μL pre-cultured bacteria.

Grow up at 37 degree for 8 hours and stored the bacteria at -70 refrigerator.

We transformed BioBricks from the 2016 igem distribution
(BBa_J23110, BBa_J23101, BBa_B0031, BBa_B0032, BBa_E1010)
Jun. 03/2016

Transformed 3 ml/μL biobrick to 100 μL DH5α competent cell by heat shock.
Result: The transformation of BBa_J23110, BBa_B0032 and BBa_E1010 was successful.
Result: The transformation of BBa_J23110, BBa_B0032 and BBa_E1010 was fail.

Jun. 04/2016

Pre-culture: 4 ml of LB (with Chloramphenicol) was inoculated with one colony from LB agar plate.

Extracted plasmid by Plasmid miniPREP Kit and confirmed plasmid size by gel electrophoresis.

Jun. 05/2016

Culture: 10 ml of LB (with Chloramphenicol) was inoculated with one colony from LB agar plate. And grow up at 37 degree for 10-12 hours.

Jun. 06/2016

Extracted the plasmid, and measure the concentration.

Stored the plasmid at -20 degree.

Jun. 07/2016

Transformed 5 ml/μL biobrick to 100 μL DH5α competent cell by heat shock.
(BBa_J23110, BBa_B0032 and BBa_E1010)
Result: The transformation of BBa_J23110, BBa_B0032 and BBa_E1010 were successful.

Jun. 08/2016

Pre-culture: 4 ml of LB (with Chloramphenicol) was inoculated with one colony from LB agar plate.

Extracted plasmid by Plasmid miniPREP Kit and confirmed plasmid size by gel electrophoresis.

Jun. 09/2016

Culture: 10 ml of LB (with Chloramphenicol) was inoculated with one colony from LB agar plate.

And grow up at 37 degree for 10-12 hours.

Jun. 10/2016

Extracted the plasmid, and measure the concentration.

Stored the plasmid at -20 degree.

Constructed pSB1C3-B0034-E1010
Jul. 01/2016

Digested pSB1C3-B0034 (SpeI,PstI) and pSB1C3-E1010 (XbaI,PstI)

Ligated E1010 (insert) to pSB1C3-B0034 (vector) and transformed the product.

Jul. 02/2016

Confirmed by colony PCR (using Prefix-F & Suffix-R as the primer).

Pre-culture: 3 ml of LB (with Ampicillin) was inoculated with one correct colony from the agar plate.

Jul. 03/2016

Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pSB1C3-B0034-E1010 is correct.

Jul. 04/2016

Culture: 10 ml of LB (with Chloramphenicol) cultured with 100 μl pre-cultured bacteria.

Grow up at 37 degree for 8 hours and stored the bacteria at -70 refrigerator.

Extracted plasmid and stored at -20 refrigerator.

Constructed pMD19T-Pcar-B0034-E1010 and pMD19T-PI-B0034-E1010
Jul. 05/2016

Digested pMD19T-Pcar, pMD19T-PI (SpeI,PstI) and pSB1C3-B0034-E1010 (XbaI,PstI)

Ligated B0034-E1010 (insert) to pMD19T-Pcar, pMD19T-PI (vector) and transformed the product.

Jul. 06/2016

Confirmed by colony PCR (using Prefix-F & Suffix-R as the primer).
Result: The length of the PCR product of pMD19T-Pcar-B0034-E1010 was incorrect.

Pre-culture: 3 ml of LB (with Chloramphenicol) was inoculated with one correct colony from the agar plate.

Jul. 07/2016

Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pMD19T-PI-B0034-E1010 was correct.

Jul. 08/2016

Culture: 10 ml of LB (with Chloramphenicol) cultured with 100 μl pre-cultured bacteria.

Grow up at 37 degree for 8 hours and stored the bacteria at -70 refrigerator.

Extracted plasmid and stored at -20 refrigerator.

Constructed pMD19T-Pcar-B0034-E1010 again
Jul. 11/2016

Digested pMD19T-Pcar (SpeI,PstI) and pSB1C3-B0034-E1010 (XbaI,PstI)

Ligated B0034-E1010 (insert) to pMD19T-Pcar (vector) and transformed the product.

Jul. 12/2016

Confirmed by colony PCR (using Prefix-F & Suffix-R as the primer).

Pre-culture: 3 ml of LB (with Chloramphenicol) was inoculated with one correct colony from the agar plate.

Jul. 13/2016

Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pMD19T-Pcar-B0034-E1010 was correct.

Jul. 14/2016

Culture: 10 ml of LB (with Ampicillin) cultured with 100 μl pre-cultured bacteria.

Grow up at 37 degree for 8 hours and stored the bacteria at -70 refrigerator.

Extracted plasmid and stored at -20 refrigerator.

Constructed pSB1C3-Pcar-B0034-E1010-TT and pSB1C3-PI-B0034-E1010-TT
Jul. 20/2016

Digested pMD19T-Pcar-B0034-RFP, pMD19T-PI-B0034-RFP (EcoRI, SpeI) and pSB1C3-B0015 (EcoRI, XbaI)

Ligated Pcar-B0034-RFP, PI-B0034-RFP (insert) to pSB1C3-B0015 (vector) and transformed the product.

Jul. 21/2016

Confirmed by colony PCR (using Prefix-F & Suffix-R as the primer).
Result: The PCR result were correct.

Pre-culture: 3 ml of LB (with Chloramphenicol) was inoculated with one correct colony from the agar plate.

Jul. 22/2016

Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of two plasmids were correct.

Jul. 23/2016

Culture: 10 ml of LB (with Chloramphenicol) cultured with 100 μl pre-cultured bacteria.

Grow up at 37 degree for 8 hours and stored the bacteria at -70 refrigerator.

Extracted plasmid and stored at -20 refrigerator.

We extracted plasmid (pMD19T-PBAD-lysis-TT) from the bacteria provided by Dr.NG
Jul. 27/2016

Pre-culture: 3 ml of LB (with Ampicillin) was inoculated with one colony from LB agar plate.

Jul. 28/2016

Culture: 10 ml of LB (with Ampicillin) was inoculated with one colony from LB agar plate. And grow up at 37 degree for 10-12 hours.

Jul. 29/2016

Extracted the plasmid, and measure the concentration.

Stored the plasmid at -20 degree.

Constructed pSB1A2-B0031-lysis-TT and pSB1A2-B0032-lysis-TT
Aug. 01/2016

PCR for lysis-TT and digested the PCR product (XbaI, PstI).

Digested pSB1A2-B0031 and pSB1A2-B0032 (SpeI, PstI).

Ligated lysis-TT to pSB1A2-B0031 and pSB1A2-B0032 and transformed the product.

Aug. 02/2016

Confirmed by colony PCR (using Prefix-F & Suffix-R as the primer).
Result: The PCR result of pSB1A2-B0031-lysis-TT was correct but pSB1A2-B0032-lysis-TT was fail.

Pre-culture: 3 ml of LB (with Ampicillin) was inoculated with one correct colony from the agar plate.

Aug. 03/2016

Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pSB1A2-B0031-lysis-TT were correct.

Aug. 04/2016

Culture: 10 ml of LB (with Chloramphenicol) cultured with 100 μl pre-cultured bacteria.

Grow up at 37 degree for 8 hours and stored the bacteria at -70 refrigerator.

Extracted plasmid and stored at -20 refrigerator.

Constructed pSB1A2-B0032-lysis-TT
Aug. 05/2016

PCR for lysis-TT and digested the PCR product (XbaI, PstI).

Digested pSB1A2-B0032 (SpeI, PstI).

Ligated lysis-TT to pSB1A2-B0032 and transformed the product.

Aug. 06/2016

Confirmed by colony PCR (using Prefix-F & Suffix-R as the primer).
Result: The PCR result was correct.

Pre-culture: 3 ml of LB (with Ampicillin) was inoculated with one correct colony from the agar plate.

Aug. 07/2016

Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pSB1A2-B0031-lysis-TT were correct.

Aug. 08/2016

Culture: 10 ml of LB (with Chloramphenicol) cultured with 100 μl pre-cultured bacteria.

Grow up at 37 degree for 8 hours and stored the bacteria at -70 refrigerator.

Extracted plasmid and stored at -20 refrigerator.

Constructed pSB1C3-J23110-B0031-lysis-TT and pSB1C3-J23110-B0032-lysis-TT
Constructed pSB1C3-J23110-B0031-lysis-TT and pSB1C3-J23110-B0032-lysis-TT
Aug. 09/2016

Digested pSB1A2-B0031-lysis-TT and pSB1A2-B0032-lysis-TT (SpeI, PstI).

Digested pSB1C3-J23101 and pSB1C3-J23110 (XbaI, PstI).

Ligated B0031-lysis-TT and B0032-lysis-TT to pSB1C3-J23101 and transformed the product.

Ligated B0031-lysis-TT and B0032-lysis-TT to pSB1C3-J23110 and transformed the product.

Aug. 10/2016

Confirmed by colony PCR (using Prefix-F & Suffix-R as the primer).
Result: The PCR result of pSB1C3-J23101-B0031-lysis-TT and pSB1C3-J23101-B0032-lysis-TT were correct.
Result: The length of the PCR product of pSB1C3-J23110-B0031-lysis-TT and pSB1C3-J23110-B0032-lysis-TT were incorrect.

Pre-culture: 3 ml of LB (with Chloramphenicol) was inoculated with one correct colony from the agar plate.

Aug. 11/2016

Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pSB1C3-J23101-B0031-lysis-TT and pSB1C3-J23101-B0032-lysis-TT were incorrect.

Aug. 12/2016

Pre-culture: 3 ml of LB (with Chloramphenicol) was inoculated with another one correct colony from the agar plate.

Aug. 13/2016

Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pSB1C3-J23101-B0031-lysis-TT and pSB1C3-J23101-B0032-lysis-TT were incorrect.

Constructed pSB1C3-J23101-B0031-lysis-TT and pSB1C3-J23101-B0032-lysis-TT again
Aug. 15/2016

Digested pSB1A2-B0031-lysis-TT and pSB1A2-B0032-lysis-TT (SpeI, PstI).

Digested pSB1C3-J23101 (XbaI, PstI).

Ligated B0031-lysis-TT and B0032-lysis-TT to pSB1C3-J23101 and transformed the product.

Aug. 16/2016

Confirmed by colony PCR (using Prefix-F & Suffix-R as the primer).
Result: The PCR result of pSB1C3-J23101-B0031-lysis-TT and pSB1C3-J23101-B0032-lysis-TT were correct.

Pre-culture: 3 ml of LB (with Chloramphenicol) was inoculated with one correct colony from the agar plate.

Aug. 17/2016

Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pSB1C3-J23101-B0031-lysis-TT and pSB1C3-J23101-B0032-lysis-TT were incorrect.

Aug. 18/2016

Pre-culture: 3 ml of LB (with Chloramphenicol) was inoculated with another one correct colony from the agar plate.

Aug. 19/2016

Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pSB1C3-J23101-B0031-lysis-TT and pSB1C3-J23101-B0032-lysis-TT were incorrect.

Constructed pMD19T-Pcar-E1010-B0015-PBAD-lysis-TT (reverse) and
pMD19T-PI-E1010-B0015-PBAD-lysis-TT (reverse)
Aug. 24/2016

PCR for Pcar-E1010-B0015 and PI-E1010-B0015.

Digested the PCR product (EcoRI, XbaI).

Digested pMD19T -PBAD-lysis-TT (EcoRI, XbaI).

Ligated Pcar-E1010-B0015 and PI-E1010-B0015 to pMD19T -PBAD-lysis-TT and transformed the product.

Aug. 25/2016

Confirmed by colony PCR (using Prefix-F & Suffix-R as the primer).
Result: The PCR results were correct.

Pre-culture: 3 ml of LB (with Ampicillin) was inoculated with one correct colony from the agar plate.

Aug. 26/2016

Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pMD19T-Pcar-E1010-B0015-PBAD-lysis-TT and pMD19T-PI-E1010-B0015-PBAD-lysis-TT were correct.

Aug. 27/2016

Culture: 10 ml of LB (with Ampicillin) cultured with 100 μl pre-cultured bacteria.

Grow up at 37 degree for 8 hours and stored the bacteria at -70 refrigerator.

Extracted plasmid and stored at -20 refrigerator.

Aug. 28/2016

Transformed plasmid (pMD19T-Pcar-E1010-B0015-PBAD-lysis-TT and pMD19T-PI-E1010-B0015-PBAD-lysis-TT) to BL21 competent cell.

Aug. 29/2016

Pre-culture: 4 ml of LB (with Ampicillin) was inoculated with one colony from the agar plate.

Aug. 30/2016

Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pMD19T-Pcar-E1010-B0015-PBAD-lysis-TT and pMD19T-PI-E1010-B0015-PBAD-lysis-TT were correct.

Aug. 31/2016

Culture: 10 ml of LB (with Ampicillin) cultured with 100 μl pre-cultured bacteria.

Grow up at 37 degree for 8 hours and stored the bacteria at -70 refrigerator.

Extracted plasmid and stored at -20 refrigerator.