Lab 3: Gel Electrophoresis and Transformation

Question 1

What was run on the gel for lab 3?

Answer 1

The restriction digest of pGFPuv and pHSG298

Question 2

What was the control that was run on the lab 3 gel?

Answer 2

Uncut pGFPuv and pHSG298. They show that the restriction digest was successful and are negative controls

Question 3

What is in the gel loading buffer?

Answer 3

Sucrose and bromophenol blue

Question 4

Why is there sucrose in the gel loading buffer?

Answer 4

Weighs down the sample so it goes into the wells and not floating around in the TBE

Question 5

Why is there bromophenol blue in the gel loading buffer?

Answer 5

Allows us to track the progress, it runs at about the same speed as a 300 bp piece of DNA

Question 6

What is transformation?

Answer 6

The uptake of DNA from outside the cell

Question 7

What were the selectable markers used for our transformation to select the colonies with the correct plasmid?

Answer 7

Antibiotic resistance and fluorescence

Question 8

Why do we use antibiotic resistance as a selectable marker?

Answer 8

Any cells without the resistance gene will die and will be removed from the analysis

Question 9

What are the 3 transformation controls?

Answer 9

Positive control, negative control, viability controls

Question 10

What were the cells plated on the positive control transformed with? What does this control tell us?

Answer 10

They were transformed with something we knew contained a kanamycin resistance gene, the pHSG298 plasmid. It tells us that the transformation was successful

Question 11

If there was no growth on the positive control plate, what could be the cause?

Answer 11

There is a problem with the competent cells that is stopping them from taking up DNA, or there was an error in the procedure

Question 12

What were the cells plated on the negative control transformed with? What does this control tell us?

Answer 12

They were transformed with only water, no DNA. It tells us there was no contamination or antibiotic resistance already present

Question 13

If there was growth on the negative control, what could be the cause?

Answer 13

Contamination or the cells were already resistant to the antibiotic

Question 14

What were the cells plated on the viability controls transformed with? What does this control tell us?

Answer 14

Viability controls were performed for the negative control (no DNA), the positive control (pHSG298) and the ligation. We do it for each because it tells us that the cells survived the transformation

Question 15

If there was no growth on the viability controls, what could be the cause?

Answer 15

The cells did not survive the transformation

Question 16

Why do we need to plate a lot of cells on the selective plates?

Answer 16

Transformation is really inefficient, so most cells won’t get any plasmids. We need to plate a lot of cells so we get cells growing on the plates

Question 17

Why do we need to perform serial dilutions for the non-selective plates?

Answer 17

Everything should grow on the LB plates, so we need to dilute the samples so we can actually count the colonies

Question 18

What is blue-white screening?

Answer 18

Using the LacZ gene to screen for an insert. If there is no insert, beta-galactosidase is functional and produces the blue product from breaking down X-gal. If there is an insert, beta-galactosidase isn’t functional and doesn’t produce the blue product, so the colonies are white

Question 19

What was the phenotype of the colonies with our recombinant plasmid?

Answer 19

Kanamycin resistant, white, and fluorescent