We got the bacteria with Biobrick from XMU.
Pre-culture: 3 ml of LB (with Chloramphenicol) was inoculated with one colony from LB agar plate.
Culture: 10 ml of LB (with Chloramphenicol) culture with 100 ml/μL pre-cultured bacteria.
And grow up at 37 degree for 10-12 hours.
Extracted the plasmid, and measured the concentration.
Store the plasmid at -20 degree.
PCR for PI & Pcar.
Result: After gel purified, the concentration of the DNA was too low to do construction.
PCR for PI & Pcar again (in big scale).
Purified The PCR product and cloned to T-vector.
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.
Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pMD19T-PI and pMD19T-Pcar were correct.
Culture: 10 ml of LB (with Ampicillin) cultured with 100 ml/μL pre-cultured bacteria.
Grow up at 37 degree for 8 hours and stored the bacteria at -70 refrigerator.
Digested pSB1C3-B0034 (SpeI,PstI) and pSB1C3-K592009 (XbaI,PstI)
Ligated K592009 (insert) to pSB1C3-B0034 (vector) and transformed the product.
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.
Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pSB1C3-B0034-K592009 was correct.
Culture: 10 ml of LB (with Chloramphenicol) cultured with 100ml/μL pre-cultured bacteria.
Grow up at 37 degree for 8 hours and stored the bacteria at -70 refrigerator.
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.
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.
Extracted plasmid and confirm plasmid size by gel electrophoresis.
Result: The size of pMD19T-Pcar-B0034-K592009 and pMD19T-PI-B0034-K592009 were correct.
Culture: 10 ml of LB (with Ampicillin) cultured with 100 ml/μL pre-cultured bacteria.
Grow up at 37 degree for 8 hours and stored the bacteria at -70 refrigerator.
Transformed 3 ml/μL biobrick to 100 ml/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.
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.
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.
Extracted the plasmid, and measure the concentration.
Stored the plasmid at -20 degree.
Transformed 5 ml/μL biobrick to 100 ml/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.
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.
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.
Extracted the plasmid, and measure the concentration.
Stored the plasmid at -20 degree.
Digested pSB1C3-B0034 (SpeI,PstI) and pSB1C3-E1010 (XbaI,PstI)
Ligated E1010 (insert) to pSB1C3-B0034 (vector) and transformed the product.
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.
Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pSB1C3-B0034-E1010 is correct.
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.
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.
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.
Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pMD19T-PI-B0034-E1010 was correct.
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.
Digested pMD19T-Pcar (SpeI,PstI) and pSB1C3-B0034-E1010 (XbaI,PstI)
Ligated B0034-E1010 (insert) to pMD19T-Pcar (vector) and transformed the product.
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.
Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pMD19T-Pcar-B0034-E1010 was correct.
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.
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.
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.
Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of two plasmids were correct.
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.
Pre-culture: 3 ml of LB (with Ampicillin) was inoculated with one colony from LB agar plate.
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.
Extracted the plasmid, and measure the concentration.
Stored the plasmid at -20 degree.
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.
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.
Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pSB1A2-B0031-lysis-TT were correct.
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.
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.
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.
Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pSB1A2-B0031-lysis-TT were correct.
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.
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.
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.
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.
Pre-culture: 3 ml of LB (with Chloramphenicol) was inoculated with another one correct colony from the agar plate.
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.
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.
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.
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.
Pre-culture: 3 ml of LB (with Chloramphenicol) was inoculated with another one correct colony from the agar plate.
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.
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.
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.
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.
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.
Transformed plasmid (pMD19T-Pcar-E1010-B0015-PBAD-lysis-TT and pMD19T-PI-E1010-B0015-PBAD-lysis-TT) to BL21 competent cell.
Pre-culture: 4 ml of LB (with Ampicillin) was inoculated with one colony from the agar plate.
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.
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.
