Team:CityU HK/notebook/lablog/project1

Week 3 (13/7~19/7)

- Primer design for leaderless tesA for Fit-coli (‘tesA) & New biobrick (‘tesA BB)

Week 4 (20/7~26/7)

- PCR of ‘tesA and ‘tesA BB

- PCR purification of ‘tesA and ‘tesA BB

- Prepare LB agar plates(‘tesA BB)

Week 5 (27/7~31/7)

- Digestion of ‘tesA BB and pSB1C3 plasmid with EcoRI & PstI restriction enzymes

- PCR purification for digested ‘tesA BB

- Sub-cloning of ‘tesA BB into pSB1C3 plasmid

- Transformation of ‘tesA BB into W3110 E.coli

- Pick colonies of the transformed E. coli with ‘tesA BB

Week 1 (1/8~2/8)

- Cell lysis of picked E. coli with ‘tesA BB

- Colonies PCR of ‘tesA BB by using VF2 & VR primer

- Send the ‘tesA BB plasmid for sequencing

Week 2 (3/8~9/8)

- 2nd PCR of ‘tesA
∵ The first PCR product of ‘tesA was degraded over a long period of storage time

- PCR purification of ‘tesA PCR product

- 1st Digestion of ‘tesA by using XbaI & PstI restriction enzyme
-> After running gel, no fragment showed in pilot ( other plasmid with the Xbal & Pstl respectively) , could not determine whether the digestion was success or not

- 2nd digestion of ‘tesA by using XbaI & PstI restriction enzyme with adding one more control pilot that is only plasmid with no enzyme
-> Still no fragment presented in pilot, we wonder whether the pilot plasmid or the restriction enzyme had problem

- 3rd digestion of lac promoter in pSB1C3 plasmid & pbad promoter in pSB1C3 plasmid by using EcoRI, XbaI, SpeI & PstI restriction enzyme
-> All of the restriction enzyme worked normally, but the pbad promoter in pSB1C3 plasmid was degraded

- 4th digestion of ‘tesA by using XbaI & PstI restriction enzyme

- Sub-cloning of‘tesA into rbs in pSB1C3 plasmid (Part: BBa_B0030)

Week 5 (27/7~31/7)

- Digestion of ‘tesA BB and pSB1C3 plasmid with EcoRI & PstI restriction enzymes

- PCR purification for digested ‘tesA BB

- Sub-cloning of ‘tesA BB into pSB1C3 plasmid

- Transformation of ‘tesA BB into W3110 E.coli

- Pick colonies of the transformed E. coli with ‘tesA BB

Week 3 (10/8~16/8)

- Cell lysis of picked E. coli with ‘tesA BB

- Colonies PCR of ‘tesA BB by using VF2 & VR primer

- Send the ‘tesA BB plasmid for sequencing

Week 2 (3/8~9/8)

- 2nd PCR of ‘tesA
∵ The first PCR product of ‘tesA was degraded over a long period of storage time

- PCR purification of ‘tesA PCR product

- 1st Digestion of ‘tesA by using XbaI & PstI restriction enzyme
-> After running gel, no fragment showed in pilot ( other plasmid with the Xbal & Pstl respectively) , could not determine whether the digestion was success or not

- 2nd digestion of ‘tesA by using XbaI & PstI restriction enzyme with adding one more control pilot that is only plasmid with no enzyme
-> Still no fragment presented in pilot, we wonder whether the pilot plasmid or the restriction enzyme had problem

- 3rd digestion of lac promoter in pSB1C3 plasmid & pbad promoter in pSB1C3 plasmid by using EcoRI, XbaI, SpeI & PstI restriction enzyme
-> All of the restriction enzyme worked normally, but the pbad promoter in pSB1C3 plasmid was degraded

- 4th digestion of ‘tesA by using XbaI & PstI restriction enzyme

- Sub-cloning of‘tesA into rbs in pSB1C3 plasmid (Part: BBa_B0030)

Week 5 (27/7~31/7)

- Digestion of ‘tesA BB and pSB1C3 plasmid with EcoRI & PstI restriction enzymes

- PCR purification for digested ‘tesA BB

- Sub-cloning of ‘tesA BB into pSB1C3 plasmid

- Transformation of ‘tesA BB into W3110 E.coli

- Pick colonies of the transformed E. coli with ‘tesA BB