Team:CityU HK/Notebook

Notebook - City University of Hong Kong 2015

Picture
Notebook
lacZY plasmid

Keys to the table:
[?/?]: Names of restriction enzyme used for digestion:; E, EcoRI; X, XbaI; S, SpeI; P, PstI
- A ribosome binding site (RBS) is linked upstream to each gene
- The host cells used for transformation were competent JM109 E. coli cells


PCR amplification of the lacZ and lacY genes.

Week 1 : May 18 – 22
Date Group 1 Group 2 Group 3 Group 4
Monday 18
==introduction==
Tuesday 19
Wednesday 20
  • PCR amplification of the BBa_S04055 lacZ gene
    		•
  • PCR amplification of the BBa_S04055 lacZ gene
    		•
  • PCR amplification of the BBa_S04055 lacY gene
    		•
  • PCR amplification of the BBa_S04055 lacY gene
    		•
    Thursday 21
  • Gel electrophoresis of the amplicon
  • lacZ PCR product purification
  • Restriction digestion with [E/P] of the vector pSB1C3 & lacZ
    		•
  • Gel electrophoresis of the amplicon
  • lacZ PCR product purification
  • Restriction digestion with [E/P] of the vector pSB1C3 & lacZ
    		•
  • Gel electrophoresis of the amplicon
  • lacY PCR product purification
  • Restriction digestion with [E/P] of the vector pSB1C3 & lacY
    		•
  • Gel electrophoresis of the amplicon
  • lacY PCR product purification
  • Restriction digestion with [E/P] of the vector pSB1C3 & lacY
    		•
    Friday 22
  • Gel purification of vector pSB1C3 [E/P] digest
  • Ligation of the [E/P] digested lacZ insert into the [E/P] digested vector pSB1C3
    		•
  • Gel purification of vector pSB1C3 [E/P] digest
  • Ligation of the [E/P] digested lacZ insert into the [E/P] digested vector pSB1C3
    		•
  • Gel purification of vector pSB1C3 [E/P] digest
  • Ligation of the [E/P] digested lacY insert into the [E/P] digested vector pSB1C3
    		•
  • Gel purification of vector pSB1C3 [E/P] digest
  • Ligation of the [E/P] digested lacY insert into the [E/P] digested vector pSB1C3
    		•
    Week 2 : May 25 – 29
    Date Group 1 Group 2 Group 3 Group 4
    Monday 25
    Tuesday 26
  • Transformation of May 22 ligation product into competent E. coli cells
    		•
  • Transformation of May 22 ligation product into competent E. coli cells
    		•
  • Transformation of May 22 ligation product into competent E. coli cells
    		•
  • Transformation of May 22 ligation product into competent E. coli cells
    		•
    Wednesday 27
  • Colony PCR of May 26 transformed cells
    		•
  • Colony PCR of May 26 transformed cells
    		•
  • Colony PCR of May 26 transformed cells
    		•
  • Colony PCR of May 26 transformed cells
    		•
    Thursday 28
  • Extraction of the plasmid pSB1C3-BBa_J23100
  • Restriction digestion with [S/P] of the vector pSB1C3-BBa_J23100
    		•
  • Extraction of the plasmid pSB1C3-BBa_J23100
  • Restriction digestion with [S/P] of the vector pSB1C3-BBa_J23100
    		•
  • Extraction of the plasmid pSB1C3-BBa_J23100
  • Restriction digestion with [S/P] of the vector pSB1C3-BBa_J23100
    		•
  • Extraction of the plasmid pSB1C3-BBa_J23100
  • Restriction digestion with [S/P] of the vector pSB1C3-BBa_J23100
    		•
    Friday 29
  • Gel purification of May 28 digest
    		•
  • Gel purification of May 28 digest
    		•
  • Gel purification of May 28 digest
    		•
  • Gel purification of May 28 digest
    		•


    Groups 1 & 3 and Groups 2 & 4 used different cloning strategies to assemble the biobrick J23100_lacZ-lacY.


    Week 2 : June 1 – 5
    Date Group 1 Group 2 Group 3 Group 4
    Monday 1
  • Extraction of the plasmid pSB1C3-lacZ (from May 26 transformed cells)
  • Restriction digestion with [X/P] of the insert lacZ
  • Gel purification
    		•
  • Extraction of the plasmid pSB1C3-lacY (from May 26 transformed cells)
  • Restriction digestion with [X/P] of the insert lacY
  • Gel purification
    		•
  • Extraction of the plasmid pSB1C3-lacZ from Gp1
  • Restriction digestion with [E/S] of the insert lacZ
  • Gel purification
    		•
  • Extraction of the plasmid pSB1C3-lacY from Gp1
  • Restriction digestion with [E/S] of the insert lacY
  • Gel purification
    		•
    Tuesday 2
  • Redo the work on June 1
    		•
  • Redo the work on June 1
    		•
  • Redo the work on June 1
    		•
  • Redo the work on June 1
    		•
    Wednesday 3
  • Ligation of Gp1 [X/P] digested lacZ insert (from June 2) into [S/P] digested vector pSB1C3-BBa_J23100 (from May 29)
  • Transformation
    		•
  • Ligation of Gp2 [E/S] digested lacZ insert (from June 2) into Gp4 [E/X] digested vector pSB1C3-lacY(from June 2)
  • Transformation
    		•
    Thursday 4
  • Colony PCR on June 3 transformed cells
    		•
  • Colony PCR on June 3 transformed cells
    		•
    Friday 5
  • Extraction of plasmid pSB1C3-BBa_J23100-lacZ (from June 3 transformed cells)
  • Restriction digestion with [S/P] of the vector pSB1C3-BBa_J23100-lacZ
  • Gel purification
    		•
  • Extraction of plasmid pSB1C3 -lacZ-lacY (from June 3 transformed cells)
  • Restriction digestion with [X/P] of the insert lacZ-lacY
  • Gel purification
    		•