Bacterial Transformation Using Pvib
BACTERIAL TRANSFORMATIONS USING PVIB
II. INTRODUCTION Transformation is the manipulation of a bacterial cell's DNA in order to alter the cell's genotype or phenotype by absorbing free DNA from its surroundings. In this lab, pVIB plasmid will be used. A plasmid is a segment of DNA that can incorporate itself into the bacterial DNA. Although is not required for growth of the bacterial cell, plasmids can provide advantages in stressful environments such as the ability to adapt as environmental changes occur. In this lab, we will obtain a better understanding of bacterial transformations using pVIB.
Hypothesis: If the bacteria transforms successfully with the pVIB, the the +plasmid (LB & AMP) will glow in the dark.
III. MATERIALS & PROCEDURES
1. Use one sterile 15-mL tube "-plasmid;" and another "+plasmid." 2. Use a sterile transfer pipet to add 250 μL of ice-cold calcium chloride each tube. 3. Place both tubes into ice. 4. Use a sterile plastic inoculating loop to transfer one large colony of E. coli cells from the starter plate to the +plasmid tube. 5. Return +plasmid tube to ice. Transfer a second mass of cells to -plasmid and do the same as +plasmid and mix thoroughly. 6. Both tubes should now be on ice. 7. Use the sterile plastic inoculating loop to transfer one loop full (10 μL) of pBLU solution (0.005 μg/μL) to the +plasmid tube. Lift the loop full of pBLU solution into cell suspension, and mix DNA. 8. Return +plasmid tube to ice, and incubate both tubes on ice for 15 minutes. While tubes are incubating, label 2 LB plate and 2 LB/Amp plates with lab group name and date.
a) Label one LB "+". (Experimental plate).
b) Label the other LB "-". (Negative control).
c) Label one LB/Amp plate "+". (Experimental plate).
d) Label the other LB/Amp plate "-". (Negative control). 9. A 15-minute incubation is followed by a "heat shock". Remove both tubes directly from ice and immediately
II. INTRODUCTION Transformation is the manipulation of a bacterial cell's DNA in order to alter the cell's genotype or phenotype by absorbing free DNA from its surroundings. In this lab, pVIB plasmid will be used. A plasmid is a segment of DNA that can incorporate itself into the bacterial DNA. Although is not required for growth of the bacterial cell, plasmids can provide advantages in stressful environments such as the ability to adapt as environmental changes occur. In this lab, we will obtain a better understanding of bacterial transformations using pVIB.
Hypothesis: If the bacteria transforms successfully with the pVIB, the the +plasmid (LB & AMP) will glow in the dark.
III. MATERIALS & PROCEDURES
1. Use one sterile 15-mL tube "-plasmid;" and another "+plasmid." 2. Use a sterile transfer pipet to add 250 μL of ice-cold calcium chloride each tube. 3. Place both tubes into ice. 4. Use a sterile plastic inoculating loop to transfer one large colony of E. coli cells from the starter plate to the +plasmid tube. 5. Return +plasmid tube to ice. Transfer a second mass of cells to -plasmid and do the same as +plasmid and mix thoroughly. 6. Both tubes should now be on ice. 7. Use the sterile plastic inoculating loop to transfer one loop full (10 μL) of pBLU solution (0.005 μg/μL) to the +plasmid tube. Lift the loop full of pBLU solution into cell suspension, and mix DNA. 8. Return +plasmid tube to ice, and incubate both tubes on ice for 15 minutes. While tubes are incubating, label 2 LB plate and 2 LB/Amp plates with lab group name and date.
a) Label one LB "+". (Experimental plate).
b) Label the other LB "-". (Negative control).
c) Label one LB/Amp plate "+". (Experimental plate).
d) Label the other LB/Amp plate "-". (Negative control). 9. A 15-minute incubation is followed by a "heat shock". Remove both tubes directly from ice and immediately